Misdiagnosis of pulmonary tuberculosis and associated factors in peripheral laboratories: a retrospective study, Addis Ababa, Ethiopia
Desalegn et al. BMC Res Notes
Misdiagnosis of pulmonary tuberculosis and associated factors in peripheral laboratories: a retrospective study, Addis Ababa, Ethiopia
Daniel Melese Desalegn 0
Kumera Terfa Kitila 0
Hanna Mekonnen Balcha 0
Chalachew Sisay Gebeyehu 0
Yohannes W. Kidan 0
Kassayenew Amare 0
Daniel Dejene 0
Merone Seifu 0
Addis Zewdie 0
Abiyot Tenna 0
Tinsae Kidanemariam Hailu 0
Boja Dufera Taddese 0
Abrham Tesfaye Bika 0
0 Addis Ababa Public Health Research and Emergency Management Core Process, Addis Ababa City Administration Health Bureau , Addis Ababa , Ethiopia
Objective: Sputum smear microscopy reading errors are likely to result in failure to detect persons with infectious TB. This study was intended to review misdiagnosis of pulmonary TB and associated factors in peripheral laboratories. Results: During the study period 1033 (10.5%) sputum smear positive and 8783 (89.5%) smear negative slides were reported by peripheral laboratories. The slides were re-read by the central referral laboratories (CRLs) as the reference standard reading. Of 1033 positive slides reported by peripheral laboratories, 25 (2.4%) were false positive. Out of 8783 smear negative slides reported by peripheral laboratories, 35 (0.4%) were false negative. The sensitivity, specificity, positive predictive value and negative predictive value of peripheral laboratories were 96.64, 99.72, 97.58, and 99.61% respectively. The peripheral laboratories and CRLs have an observed agreement (Po) of 0.9939. Of 135 peripheral laboratories, 93 (68.9%) read negative and positive slides correctly, 49 (36.3%) did not have lens cleaning tissue papers, 11 (8.1%) lacked frosted slides, and 14 (10.4%) had shortage of reagents. As conclusions, the peripheral laboratories and CRLs had high agreement for sputum smear microscopy reading. However, a few TB cases were misdiagnosed despite having the disease; these individuals might continue to spread the infection in the community.
External quality assessment; ZN sputum smear microscopy; Addis Ababa
Laboratory services continue playing critical role in
diagnosing TB and treatment monitoring [
]. However, it
requires strong quality assurance (QA) systems be in
]. In contrary, reading errors in ZN-stained
sputum smear microscopy are likely to result in
failure to detect persons with infectious TB, consequently
continues to spread infection in the community, or may
unnecessary treated for non TB cases [
in adequately training, erratic reagent, supplies, and poor
equipment maintenance inflate the problems.
Accurate and reliable TB microscopy result relies on
external quality assessment (EQA) programs that
support, train, and monitor testing performance of
individual laboratories [
]. External quality assessment
comprises proficiency testing, blind-rechecking of
samples and on-site evaluation [
]. In study setting,
blindrechecking programs is crucial EQA method in assessing
sputum smear microscopy reading performance of the
]. It provides an opportunity to assess
quality performance elements including specimen
quality, smear size and thickness, and quality of staining. All
these information may be very useful to assessing
possible reasons for false positive or false negative results, and
implementing plans for retraining and corrective action
3, 9, 10
Information concerning the performance of EQA in
private and public laboratories in Addis Ababa is very
limited; hence we reviewed 135 public and private health
facilities’ ZN sputum smear microscopy EQA
performance, to determine misdiagnosis of pulmonary TB
cases and associated factors in private and public health
facilities in Addis Ababa, Ethiopia.
Retrospective review of EQA records in 135 public and
private laboratories in Addis Ababa Ethiopia was
conducted, from October, 2014 to March, 2016. These
peripheral laboratories provide TB diagnostic services
and they participate in ZN smear microscopy Regional
External Quality Assurance Scheme (REQAS). This study
compared the results of peripheral laboratories’
ZNstained sputum smear microscopy reading performances
with CRLs as a gold standard.
One hundred thirty five peripheral laboratories (45
privates and 90 public) providing TB diagnostic service to
patients in Addis Ababa were considered for the current
Data sources and sampling
Data were collected from 135 health facilities in Addis
Ababa. The health facilities’ ZN-stained sputum smear
microscopy EQA performances and onsite EQA records
were used as data source.
Inclusion and exclusion criteria
All peripheral laboratories having complete EQA
performance records in the CRLs data storage, from the
years 2014–2016 were included. However, TB
laboratory results performed by other than ZN technique were
Data management and statistical analysis
The collected data were checked for completeness and
consistency and entered into using Epi-info software.
The data were coded and analysed using SPSS version
20.0 (SPSS Inc. Chicago, USA) software. ZN-stained
sputum smear microscopy reading agreement between
the peripheral microscopy centers and the CRLs were
measured by using Kappa (K). Inter-observer variability
was assessed on the basis of κ-values, with kappa
coefficient: 0.81–1 = almost perfect, 0.61–0.8 = substantial,
0.41–0.60 = moderate, 0.21–0.40 = fair, 0.01–0.20 = slight
and ≤ 0 = poor agreement between slide readers [
Sensitivity, specificity, PPV and NPV of the peripheral
laboratories were calculated against the final readings of
the CRLs as a gold standard. Bivariate and Multivariate
logistic regression model using odds ratio (OR) at 95%
confidence interval (CI) were calculated. P value less than
0.05 were taken as statistically significant.
Data quality assurances
Before extracting data from records, data collectors were
adequately trained and the collected data were checked
for completeness by data collectors and authors. Data
collection process was supervised and monitored by the
Central referral laboratories (CRLs): Regional and
intermediate level (Hospital) laboratories serving as
ZN smear microscopy a quality assurance (REQAS)
centers for peripheral laboratories.
Peripheral laboratories: Laboratories located at a
Health Center or private Hospital and different level
of Clinics, providing ZN smear microscopy
Discordant slides: Positive slides read as negative or
ZN‑stained sputum smears microscopy reading agreement
During the study period 135 peripheral laboratories were
involved in REQAS. Among these, 45 (33.3%) were
private and the remaining 90 (66.7%) public health facilities.
Of 135 peripheral laboratories, 93 (68.9%) correctly read
negative and positive sputum smear slides. The
remaining 42 (31.1%) laboratories misread at least one positive
sputum smear slides as negative or vice versa. Out of
42 health peripheral laboratories reported false
reading results, 23 (54.8%) were public and the remaining 19
(45.2%) were private’ laboratories. However, there was
no statistically significant difference in sputum smear
microscopy false reading results between public and
private laboratories (P value > 0.05).
Of 9816 ZN-stained sputum smear slides were
collected from the peripheral laboratories for re-checking,
1033 (10.5%) were reported as positive, and the
reaming 8783 (89.5%) were reported as negative by peripheral
laboratories. The slides were re-read by the CRLs, which
were considered as the reference standard reading. Of
1033 sputum smear slides reported as positive by
peripheral laboratories, 1008 (97.6%) were true positive, the
remaining 25 (2.4%) were false positive. Out of 8783
sputum smear slides reported as negative by peripheral
laboratories, 8748 (99.6%) were true negative, the remaining
35 (0.4%) were false negative. The overall discordant rate
between peripheral laboratories and CRLs was 60 (0.61%)
The study has shown an observed agreement (Po) of
0.9939, expected agreement (Pe) of 0.8109 and calculated
kappa value was 0.97, which indicated almost perfect
agreement. The sensitivity, specificity, PPV and NPV of
peripheral laboratories in ZN-stained sputum reading
was 96.64, 99.72, 97.58 and 99.6% respectively.
None of the peripheral laboratories assessed met sputum
smear quality standards. Of 9816 collected sputum smear
slides from the peripheral laboratories, 3475 (35.4%)
specimen quality, 3316 (33.8%) evenness, 3004 (30.6%)
smear size and 2817 (28.7%) labeling were found as poor.
Moreover, 3846 (39.2%) smears were prepared with
inappropriate thickness, and 3029 (30.9%) smears were
unacceptable staining background cleanness. Compared to
the performance of public and private laboratories for
each of the quality indices, of 3168 sputum smear slides
collected from the private laboratories, 1163 (36.7%)
specimen quality, 1042 (32.9%) evenness, 991 (31.3%)
smear size, 916 (28.9%) labeling, 1291 (40.8%) thickness,
and 1039 (32.8%) staining background were
unacceptable. Of 6648 slides collected from the public laboratories,
2312 (34.8%) specimen quality, 2274 (34.2%) evenness,
2013 (30.3%) smear size and 1901 (28.6%) labeling, 2552
(38.4%) thickness, and 2053 (30.9%) staining background
were found as poor.
Tuberculosis laboratory facilities and infrastructures
On-site evaluation of EQA was conducted in 135
peripheral laboratories to assess the status of TB
laboratory infrastructures and factors affecting the quality of
ZN-stained sputum microscopy diagnosis. The entire
assessed peripheral laboratories had functional light
microscope, 104 (77.0%) had separate work station for
smearing, staining, microscopy examination and
recording. Of the total TB laboratories assessed, 79 (58.5%) had
clean, separate and ventilated TB laboratory room, 127
(94.1%) had regular water supply and 131 (97.0%) had
back up electric power supply (Table 2).
Shortage of supplies including all type of reagents
(Carbol-fuchsin, methylene blue and Acid alcohol), lens
tissue; frosted slides, internal quality control (IQC)
reagents were identified in most peripheral laboratories. Of
135 peripheral laboratories, 49 (36.3%) did not have lens
cleaning tissue papers, 11 (8.1%) lacked frosted slides, 14
(10.4%) had shortage of ZN-staining reagents, and nearer
to half (42.2%) did not filter 1% carbol-fuchsin during
staining of sputum smears. However, all peripheral
laboratories used the appropriate personal protective
equipment (PPE) when performing TB laboratory procedures,
and they used incinerator to dispose sputum, and other
sputum contaminated solid wastes (Table 2).
There were gaps in having valid internal quality control
materials in all assessed laboratories. Among these, 38
(28.1%) laboratories prepare smears from known positive
and negative sputum as internal quality control to check
reagents quality before staining patient sample. About 59
(43.7%) did not check sputum quality before processing.
Among 135 visited laboratories, 109 (80.7%) had
standard TB laboratory request form and result log book, 107
(79.3%) had TB national guideline; manual, SOPs and
other reference materials for smear microscopy
technique. More than half (76.3%) of the peripheral
laboratories’ staffs members were trained on sputum smear
microscopy (Table 2). From those who took training, 73
(70.9%) were from public health facilities.
Factors affecting sputum smear microscopic reading
In binary logistic regression, false reading of sputum
smear microscopy had significant association with
checking the quality of sputum before smear preparation,
having separate TB laboratory room and filtering
carbolfuchsin before staining sputum smear (P value < 0.05).
However, in multivariate logistic regression analysis, false
reading had no significant association with these factors
(P value > 0.05) (Table 3).
In the current study, sputum smear microscopy reading
agreements between the peripheral laboratories with the
CRLs were evaluated, using CRLs as reference standard.
The sensitivity, specificity, PPV and NPV of the
peripheral laboratories were 96.64, 99.72, 97.58 and 99.61%,
respectively, which is comparable to 95, 99.7, 93.3 and
99.7% respectively reported in Ethiopia and 91.3, 98.9,
92.2 and 98.8% respectively reported in Argentine [
]. In contrast, it is higher than others studies [
In present study overall sputum smear reading agreement
Having functioning incinerator
between the peripheral diagnostic laboratories and CRLs
was 99.4%, which is comparable to 99.5% reported in
west Amhara Region  and 99.7%, reported Lima, Peru
]. However, it is higher compared to 96.8% reported
in south Ethiopia [
] and 87% reported in Addis Ababa
] and 89.2% report in Tanzania [
]. Although, the
proportion of sputum smear microscopy reading
agreement of 99.4% achieved is higher than acceptable
performance of > 80% [
7, 10, 21
], still disagreement of 0.6% may
indicate a need to improve the quality of TB microscopic
In this study overall discordant (false reading) results
of sputum microscopy reported by the peripheral
laboratories was 60 (0.6%), which is lower than the
previous reports of 7.8% in Argentine, 3.5% in west Amhara
Region, 3.2% in southern Ethiopia, 3.3% in India, and
5.5% in eastern part of Ethiopia [
13, 17, 19, 22, 23
]. It is
higher than study reported 0.2% in Ethiopia and 0.3% in
]. These variations might be due to sputum
smear microscopy reading is highly dependent on the
training, diligence of microscopist and laboratory
supplies. The other possible reason, unfiltered fuchsin
crystals reagents, weak decoloration of AFB might affect the
quality of ZN smear microscopy reading and lead to false
reading results [
16, 20, 26
In the present study specimen quality, evenness,
smears size, inappropriate labeling, thickness, and smears
with unacceptable staining background cleanness were
35.4, 33.8, 30.6, 28.7, 39.2 and 31.5% respectively, which
is relatively lower than other studies [
container, sample transport condition, specimen
quality, labeling, smearing, and staining technique including
failure to filter carbol fuchsin are some of the factors that
compromise ZN sputum smears quality performance
In the current study 79 (58.5%) of the peripheral
laboratories having clean, separate and ventilated TB
laboratory room, which is higher than 18.2–42% previously
reported in other studies [
15, 16, 27, 28
]. Unventilated TB
laboratory room can create unfavorable working
environment, which might affect the overall quality of sputum
smear microscopy services from pre-analytical to post
analytical phases. In addition, this may create a chance of
cross contamination [
Regarding TB laboratory safety issue, all the
peripheral laboratories used the appropriate PPE and disposed
sputum and other sputum contaminated solid wastes
appropriately. This finding was higher when compared
with study conducted in Malawi, which reported most
hospitals’ laboratories didn’t wear white coat, face mark,
protective apron and soap for washing hand [
difference might be due to availability of PPE, professionals’
attitude towards PPE. A poor laboratory safety practice
does not only put the laboratory workers at risk of
infection, but also the patients and any other person accessing
the laboratory services.
In the present study peripheral laboratories and CRLs
had high agreement for ZN sputum smear microscopy
reading. However, a few TB cases were misdiagnosed
despite having the disease; these individuals might
continue to spread the infection in the community. This
indicates the needs to improve ZN-stain sputum smear
microscopy reading to attain the end TB strategy.
Limitations of the study
• Results of this study depend only on the TB-EQA
record review. Therefore, not illustrate providers
AFB: acid fast bacillus (or bacilli); AOR: adjusted odds ratio; CI: confidence
interval; CRLs: central referral laboratories; EQA: external quality assessment;
NPV: negative predictive value; COR: crude odds ratio; SPSS: Statistical Package
for Social Sciences; PPV: positive predictive value; TB: tuberculosis; WHO: World
Health Organization; ZN: Ziehl–Neelsen stain; REQAS: Regional External Quality
DMD designed the study, develops the proposal, lead data collection, analysis
and writing up the result. BD, and AT critically review and made
progressive suggestions in the study. KT, HM, CS, YWK, KA, DD, MS, AZ, AT, TKH were
participated in data collection, analysis, interpretation, and write up the result.
All authors read and approved the final manuscript.
1 Addis Ababa Public Health Research and Emergency Management Core
Process, Addis Ababa City Administration Health Bureau, Addis Ababa, Ethiopia.
2 Ethiopian Public Health Institute (EPHI), Addis Ababa, Ethiopia.
The authors would like to acknowledge Addis Ababa City Administration
health Bureau Pubic Health research and Emergency management core
processes. We sincerely thank respective health facilities for their cooperation. Our
thanks also extend to all of the CRLs laboratory staffs for their commitment
during slides rechecking.
The authors declare that they have no competing interests.
Availability of data and materials
All data were presented in the result parts of the manuscript.
Consent for publish
Ethical approval and consent to participate
Ethical approval was obtained from Ethics committee of Addis Ababa City
Administration Health Bureau Public Health Research and Emergency
management core processes.
The authors had no funding support or funding to report.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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