# Front-Loading Sputum Microscopy Services: An Opportunity   to Optimise Smear-Based Case Detection of Tuberculosis in High Prevalence Countries

Authors: Andy Ramsay; Mohammed Ahmed Yassin; Alexis Cambanis; Susumu Hirao; Ahmad Almotawa; Mohamed Gammo; Lovett Lawson; Izabel Arbide; Nasher Al-Aghbari; Najla Al-Sonboli; Jeevan Bahadur Sherchand; Punita Gauchan; Luis Eduardo Cuevas
Journal: Journal of Tropical Medicine
 (2009)

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## Abstract

Setting. Ethiopia, Nepal, Nigeria, and Yemen. Objective. To reduce the time to complete
sputum microscopy.
Design. Cross-sectional surveys enrolling 923 patients with chronic cough in the 4 countries and using similar protocols. Spot-morning-spot sputum specimens were collected.  An additional sputum specimen (Xspot) was collected one hour after the first, and the yields of the first two or the three specimens collected as spot-morning-spot or
spot-Xspot-morning were compared.  Results.  216 patients had ≥
one positive smear.  210 (97%) were identified by the
spot-morning-spot, and 210 (97%) were identified  by the spot-Xspot-morning specimens, with 203 and 200 identified by the first 2 specimens of each approach, respectively.  Neither difference was significant. Conclusions. The time to complete smear microscopy could be reduced.


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## Body

## 1. Introduction

New
diagnostics  for pulmonary tuberculosis (PTB) that are more  sensitive than
sputum smear microscopy and suitable for primary health care (PHC) services in
low- and middle-income countries (LMICs) are urgently needed. Although several
promising new diagnostics are under development, they are unlikely to become
widely available at the lower levels of LMIC health services in the near future
[1].  In the meantime, TB case detection must be
improved through  the optimal use of existing diagnostic tools.  The optimisation of sputum microscopy
services, often the only TB diagnostic services possible at PHC level  in LMICs,
is urgently needed [2–6].  Smear microscopy has several limitations,
including poor sensitivity, being labour intensive, and requiring skilled
microscopists. Furthermore, the need to collect serial sputum specimens over
multiple patient visits results in a protracted diagnostic process with high
rates of patient drop-out  [7, 8].Recent
studies examining the yield of serial sputum specimens, usually collected asspot-morning-spot, have reported that
the majority of patients with smear-positive PTB are identified by the first
two sputum specimens [2],
and the World Health Organization (WHO) has recently changed its policy in this
respect,  reducing the minimum number of
sputum specimens examined for each patient from three to two [9].
This will result in reduced laboratory workloads in many settings, with the
potential of improving the quality of sputum microscopy [10].
Case detection may thus be expected to increase in locations where the number
of new cases detected through improved microscopy quality exceeds the 2% to 5%
of cases missed by not examining the third specimen [5].The
policy changes do not, however, specify the timing for the collection of the
two specimens.  If specimens were
collected at the time of consultation (1ston-the-spot) and the
morning of the following day (morning sample), the spot-morning specimens would still require a minimum of two visits, which is the minimum
required by the spot-morning-spot scheme currently used in most
diagnostic centres of LMICs.  In addition,
the spot-morning and spot-morning-spot schemes still examine a
substantial proportion of samples the second day of the diagnostic
process.  If the process could be
“front-loaded”, that is, if all or the majority of sputum collections were
conducted the first day of the diagnostic process, this may reduce the number
of visits required and reduce patient drop-out, particularly if results could
be made available the same day.This
study describes the yield of a front-loaded diagnostic scheme, in which an additionalon-the-spot specimen (referred here as the Xspot) is collected
one hour after the first spot specimen.
We hypothesised that the yield of this specimen is similar to the yield
of other specimens collected on the spot, and that the overall yields of the spot-Xspot-morning and the standard spot-morning-spot schemes are similar.  In addition, the study explores whether the first
two specimens collected identify the majority of smear-positive patients.


## 2. Materials and Methods

Four separate studies were
conducted in Ethiopia, Nepal, Nigeria, and Yemen using similar study designs.
All individuals 15 years of age or older with cough for  ≥3 weeks
were invited to participate and were
enrolled consecutively at the time of presentation to the health
services and after obtaining informed consent. The services in all four
settings were busy outpatient clinics of district hospitals that were
integrated with the National TB Control Programme of the country.  Individuals either self-presented to these
clinics or had been referred from peripheral health centres for assessment. Patients
were requested to submit 3 sputum specimens asspot-morning-spot (the
standard approach), and an additional specimen was collected one hour after the
first (the Xspot) on the first day of consultation (Figure 1).  All smears were stained using the hot
Ziehl-Neelsen method, were read blindly by trained laboratory technicians, and
graded using the WHO/IUATLD system. In accordance with the  recent WHO policy
changes, all smears with ≥1 acid fast bacillus /100 high power fields were considered positive.  The main difference in study design between
the four sites was that all morning specimens collected in Nepal and
Yemen were cultured on solid egg-based media to allow for the calculation of
sensitivity and specificity when considering culture as the reference standard.
The four study sites had internal quality control procedures in place.  External quality assessment (EQA) of smear
microscopy was conducted by the Liverpool School of Tropical Medicine (LSTM),
UK.  For all 4 sites, there was >98% agreement
between the results of the study microscopists and those of the controllers at
LSTM.Figure 1
Sputum samples collected and analysed for each scheme.Data were analysed to describe the
yield of single smears and the cumulative yields of the standard (spot-morning-spot)
versus the front-loaded (spot-Xspot-morning) schemes and of the two
smear spot-morning versus the front-loaded spot-Xspot schemes.  Proportions were described as
percentages and 95% confidence intervals (95%CI).  A number of patients (62, 7%) did not have
complete sets of sputum specimens for analysis.
These patients were included in the analysis for the additional yield,
and missing smears were considered as negative.
As the schemes were not independent, comparisons of marginal
proportions were made using matched McNemar tests.  These
included comparisons of the extra yield of the third sputum for each scheme;
the yield of the “two” sample schemes and the comparison of the dropouts if
patients were examined by the standard and frontloaded schemes. The extra yield
of the third sputum for each scheme was summarised in 3×3  tables using the categories “positive on at least one of the
first two samples”, “positive on the  third sample only”, and “negative on all
samples” and stratified by study setting.
As this analysis required complete sets of data, the 62 (7%) patients
with missing sputum samples were excluded from this analysis. The yield for the
“two” sample schemes  was
also compared with 2×2 tables with the indicators for each scheme being “positive
on at least one of the two specimens” and “negative on both specimens”. Patients
with one of the two specimens missing (36, 4%) were classified as negative if the
specimen available was negative.  McNemar
and Stuart MaxWell tests  for marginal heterogeneity were used for comparison of the
two and three specimen schemes, respectively, stratified by study site.Partial
results of the studies in Ethiopia and Nigeria, which described the yield of
two smears collected asspot and Xspot in a single day, have been
reported previously [3, 4].  However, this report includes the yields of
the spot-morning-spot and spot-Xspot-morning schemes from these
sites as well as the data from two additional sites conducting studies with
similar design approaches.Ethical approval for the study protocols was obtained from the
research ethics committees of the Liverpool School of Tropical Medicine and the
Institutional Review Boards of the participating institutions in Ethiopia,
Nepal, Nigeria, and Yemen.


## 3. Results

A total of 923 consecutive patients
were recruited.  Of these, 243 were
enrolled in Ethiopia, 206 in Nepal, 224 in Nigeria, and 250 in Yemen.  Two hundred and sixteen (23%) of the 923
patients had one or more positive smears. Thefirst-spot, Xspot, morning, and second-spot specimens were graded positive in 181 (20%), 186 (20%),
185 (20%), and 176 (19%) patients, respectively, as shown in Table 1.  In addition to the 181 patients identified by
the first-spot, 22 (10%) and 7 (3%) additional patients were detected by
the morning and second-spot specimens, respectively, resulting in
a cumulative yield for the spot-morning-spot scheme of 210 (23%)
patients. In comparison, the Xspot and morning specimens of the
front-loaded approach detected 18 (8%) and 11 (5%) additional patients, also
resulting in a cumulative yield for the spot-Xspot-morning scheme of 210
(23%) patients. The yield of the third smear was the same in both approaches,
independently on whether this was thesecond spot or the morning specimen (P  >.5). A
comparison of the yield of the third smear for samples collected using the
standard (spot-morning-spot) and the
front-loading (spot-Xspot-morning)
approaches by country is shown in Table 2.
Although there were a few discrepant results in the yield of the third
specimen across the schemes, the direction of these discrepancies varied across
study sites, and the discrepancies in each study site or combined were not
statistically significant.Table 1
Incremental yield of serial smears collected as spot-morning-spot or
spot-Xspot-morning specimens.
CountryN*≥1 pos. smear,First spot pos.Standard approachFront-loaded approachN (%)N (%)Incremental yield,N (%)N (%) detectedIncremental yield,N (%)N (%) detectedMorningSecond spotXspotMorningEthiopia24352 (21)45 (87)4 (8)2 (4)51(98)4 (8)3 (6)52 (100)Nepal20655 (25)39 (71)7 (13)5 (9)51 (93)5 (9)6 (11)50 (91)Nigeria22448 (21)45 (94)3 (6)048 (100)2 (4)1 (2)48 (100)Yemen25061 (24)52 (85)8 (13)060 (98)7 (12)1 (2)60 (98)All923216 (23)181 (84)22 (10)7 (3)210 (97)18 (8)11 (5)210 (97)N* = number; pos = positive; Xspot = Extra spot
collected one hour after the first spot sputum specimen.Table 2
Yield of the third smear for samples
collected using the standard (spot-morning-spot) and the front-loading
(spot-spot-morning) approaches by country.  62 patients (15 from Yemen, 44 from Nepal, and 3 from Ethiopia)
were excluded as they did not submit the third specimen.
Standard
approachFront-loading
approachP*Yemen (N = 235)Pos on 1st two smearsOnly 3rd smear posAll negative.14Pos
on 1st two smears5640Only 3rd smear pos000All
negative00175Nepal (N = 162).76Pos
on 1st two smears4060Pos on the 3rd smear305Negative in all30105Nigeria (N = 224).61Pos
on 1st two smears4710Pos
on the 3rd smear000Negative in all00176Ethiopia (N = 240).55Pos on 1st two smears4630Pos
on the 3rd smear200Negative in all10188All sites (N = 861).58Pos
on 1st two smears189140Pos
on the 3rd smear505Negative
in all40644S*tuart-MaxWell test, (Marginal Heterogeneity).The first two (spot-morning)
smears of the spot-morning-spot approach identified 203 (97%) smear-positive patients.  These two smears, therefore, would have
missed 7 (3%, 95%CI 1%–6%) of the cases identified by the three smears, as
shown in Table 1.  The first two (spot-Xspot) smears of the spot-spot-morning approach identified
199 (95%, 91%–97%) smear-positive patients.
These two spot smears, therefore, would have missed 11 cases (5%, 95%CI
3%–9%) identified by the three smears.  A
matched comparison of the yield of two smears collected using the standard (spot-morning) and the front-loading (spot-Xspot) approaches stratified by
country is shown in Table 3. Again, the proportion of cases missed by the spot-morning and the spot-spot smears is not statistically different.Table 3
Yield of two smears collected using the standard
(spot-morning) and the frontloading (spot-Xspot) approaches by country.
Standard
approachFrontloading
approachP*YemenPositiveNegativeAll.18Positive56460Negative1189190All57193250Nepal.53Positive40646Negative4156160All44162206Nigeria.32Positive47148Negative0176176All47177224Ethiopia1.0Positive46349Negative3191194All49194243All sites.58Positive18914203Negative8712720All197726923M*cNemar test.Fifty-one (25%) of the 206 patients in Nepal and 72
(29%) of the 250 patients in Yemen had a positive TB culture. Thirty-nine and
37 of the 51 culture-positive patients in Nepal were identified by the standard
and the front-loaded schemes, resulting in a sensitivity of 76% [95%CI 63%–87%] and 73%
[59%–83%], respectively.  In Yemen, 51
and 52 of the 72 culture-positive patients were identified by the standard and
front-loaded schemes, respectively, (sensitivity 71% [60%–80%] and 72% [61%–82%]).  There were no statistical differences between
the sensitivities of the standard and front-loaded approaches.The sensitivity of two smears collected asspot-morning would be 69% (35 of 51
culture positive patients) in Nepal and 71% (51 of 72) in Yemen.  In comparison, the sensitivity of the two
smears collected as spot-Xspot would
be 67% (34 of 51) in Nepal and 67% (48 of 72) in Yemen.  These differences are not statistically
significant (P >.4 for both).


## 4. Discussion

Much progress has been made in recent years in the development of new
diagnostics for TB. However, few of the newer diagnostic technologies are
suitable for use outside of reference laboratories in the public health
services of LMICs. TB
control in resource-poor, high-prevalence settings, therefore, will continue to
rely upon sputum smear microscopy until frontline services gain access to the
new technologies. Tuberculosis affects disproportionately indigent
populations who seek health services in areas with limited resources [11], and the costs
incurred by patients can be prohibitively high, even when services are provided
free of charge [12].  Individuals need to attend health facilities
on several occasions to see a clinician, submit sputum specimens, receive
results, and be put on treatment, and these visits may incur loss of earnings
and require repeated travel, purchase of food, and accommodation [13].  A significant proportion of people undergoing
investigations for TB drop-out of the smear diagnostic pathway, and these
patients are more likely to be the poor. Although few studies report the
proportion of patients who drop out during the diagnosis of TB in LMIC, 13% of TB
suspects in India [14],
15% and 37% of smear-positive patients in rural [8],
and urban Malawi [15]
dropout of the process, and unusually, the diagnostic dropout rate can be as
high as 95% [16].  Failing to complete the
diagnosis, therefore, is a major obstacle to access treatment in these settings
[8].There is increasing interest in equity in health services, as typified by the
interim report of the WHO Commission on Social Determinants of Health.  Primary health care, once again, plays a
central role in WHO’s current agenda, and the development of equitable diagnostic
services is, therefore, paramount to access treatment [17].  The investigation of suspected PTB in many
low-prevalence countries is based on the examination of serial morning specimens because these specimens have been associated with a significant
additional yield of patients.  The
proportion of patients who drop out in these settings, however, is
insignificant, and the spot-morning-spot scheme was developed in
the 1950s and 60s in response to the need to reduce the number of visits in
high-prevalence areas, where patients often abandoned the diagnostic process.  Although it was widely accepted that
overnight specimens were more likely to contain more bacilli, it was also
acknowledged that in less favourable circumstances, it was more practical to
obtain specimens at the time the patient was attending the service [18].  Two of the morning specimens were replaced by
spot specimens, and shorter schemes that required a reduced number of visits
were developed. Although this was a remarkable improvement, the continued need
for multiple visits was still a hindrance for many patients.It is now recognised that the high
bacillary threshold for defining a smear as positive and the requirement to
obtain at least one confirmatory smear have unnecessarily reduced the sensitivity of the
test in the detection of smear positive cases.
These requirements also resulted in many laboratory services being overwhelmed
and leaving insufficient time for the examination of smears.  The recent WHO policy changes reduce the
smear microscopy thresholds [9]
and the minimum number of specimens to be examined. These changes will reduce
workload, particularly important in areas, where skilled human resources are
limited, potentially increasing case detection through allowing more time to
examine the smears [10].
These policy changes might be associated with larger gains in case detection if
the timing of sputum specimen collection and examination were more convenient
for patients, particularly poor patients, and help to reduce drop-out.This study shows that thespot-spot-morning and spot-morning-spot schemes have similar yields.  This
indicates that front-loaded TB diagnostic services (whether based on the
examination of two or three specimens) are feasible and would not be associated
with significantly less yield than the equivalent standard approach.  Both two-smear strategies would miss about 3%–5% of the
patients identified by the three-specimen strategy, as suggested by a previous
systematic review [5, 19].  These losses are likely to be compensated by
increased quality of microscopy and lower drop-out rates of patients.  In addition, programmes
where  ≥10% of patients fail to return for the second day of diagnosis would
identify similar numbers using 2 smears the first day of consultation than 2 or
3 smears collected over two or more days. Countries adopting two-smear schemes,
therefore, may consider collecting and examining the specimens in a single day
to shorten the time required for diagnosis.The development
of diagnostic approaches that are responsive to the needs of the population may
be feasible, and larger studies are urgently required to validate the findings
of this study under operational conditions.
If the findings of this study are confirmed, smear microscopy services should be
front loaded in the interests of equity and improved TB control.


## 5. Summary

The diagnosis of tuberculosis in
high-burden settings relies on sputum smear microscopy and requires multiple
patients’ visits to the health facilities.
This approach could be improved if most specimens were collected the
first day of consultation.  This study
reports the smear microscopy findings of 923 adults with chronic cough participating in four cross-sectional surveysin Ethiopia,
Nepal, Nigeria, and Yemen.  Sputum
specimens were collected as spot-morning-spot plus one additional specimen one
hour after the first spot (X-spot).  The yield of two
(spot-Xspot or spot-morning) or three (spot-morning-spot
or spot-X-spot-morning) specimens was
compared.   216 patients had ≥ one positive smear.  Of these, 210 (97%) were identified by the spot-morning-spot,
and 210
(97%) were identified by the spot-Xspot-morning specimens.  Spot-morning identified 203 and spot-Xspot
specimens 200 patients, respectively, (P >.1). The time, number of
visits and patients’ costs to complete smear microscopy could be reduced by
frontloading the collection of sputum specimens.



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