ORIGINAL ARTICLE
A COMPARATIVE STUDY OF SPUTUM SMEARS BY ZIEHL NEELSEN
STAINING VERSUS FLUORESCENT STAINING IN THE DIAGNOSIS
OF PULMONARY TUBERCULOSIS IN PATIENTS SUFFERING WITH
COUGH FOR 2 WEEKS OR MORE
R. Ramakrishna1, Chundari Bharavi2
HOW TO CITE THIS ARTICLE:
R. Ramakrishna, Chundari Bharavi. ”A Comparative Study of Sputum Smears by Ziehl Neelsen Staining
Versus Fluorescent Staining in the Diagnosis of Pulmonary Tuberculosis in Patients Suffering with Cough for
2 weeks or more”. Journal of Evidence based Medicine and Healthcare; Volume 2, Issue 11, March 16,
2015; Page: 1663-1674.
ABSTRACT: 1. Total study population consists of 200 patients. The results of 200 patients after
performing ZN staining and AR staining have been analyzed. 2. The study population consists of
130 males and 70 females. Males were more than females in this study population. 3. The study
group varied in age between 19-88 years of which 130 were Males and 70 were Females. The
youngest patient was aged 19years and the oldest 88 years. 4. The mean age of the study
population was 28.57. The predominant age group involved were 19-28 years group and 59 to 68
years group followed by 49 to 58 years group. The least involved group was 79-88 years age
group. 5. Out of 58 sputum smear positive patients, 42 patients were males, 16 patients were
females with a male to female ratio of 2.62:1.6. The findings showed that Fluorescent staining
and ZN staining preparation each showed positive for AFB in 58(29%) out of 200 and 40(20%)
out of 200 patients respectively. The FM showed more sputum smear positivity than ZN
Microscopy. 18 patients were positive by AR Staining alone and negative by ZN staining. 7. Out of
the 58 sputum positive patients, right side was involved in 25 patients chest x-rays, left side in 19
patients chest x-rays, bilateral in 12 patients chest x-rays and normal in 2 patients. Right side
was predominantly involved than Left side. 8. Number of patients with spot sputum smears
negative and early morning sputum smears positive or scanty are 13(22.4%). 9. Difference in
Grading between spot sputum smear and early morning sputum smear occurred in 27 patients
(46.5%) of which early morning sample is of higher grade than spot sputum sample. 10. Number
of HIV positive patients was 10. Out of which 5 patients were sputum smear positive and 5
patients were sputum smear negative.
KEYWORDS: AFB - Acid Fast Bacilli, AIDS - Acquired immune deficiency Syndrome, AR Auramine o rhodamine stain, ATT - Anti Tuberculosis Therapy, ART - Anti Retroviral Therapy,
DOTS - Directly observed Treatment short course Therapy, HIV - Human immunodeficiency virus.
INTRODUCTION: Hageman 65 in 1937 introduced fluorescent microscopy in the examination of
acid fast bacilli. Matthai first developed the present staining technique of using combination of
auramine and rhodamine for acid fast bacilli in 1950. Since then large number of reports have
been published regarding its efficacy. In 1965, a detailed study by the microbiology and
pathology departments of Veterans Administration center, Wisconsin, U.S.A. established the
supremacy of Fluorescent Microscopy (FM) over routine Ziehl Neelsen method in sputum and
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ORIGINAL ARTICLE
tissue specimen training. In our country in 1960’s the National Tuberculosis Institute,(1) Bangalore
had done some work on FM and abandoned the procedure due to its operational difficulties,
equipment cost, maintenance cost and cost of imported stains.
WHO1 has recommended that the maximum number of ZN smears examined by a
microscopist in a day should not exceed 201. If more than this number of examinations is
attempted, faulty readings will occur. The proficiency in reading the ZN smears can only be
maintained by examining at least 10-15 ZN smears per week that is an average of 2-3 smears per
day. The field seen in Fluorescent microscopy6 is 0.34 mm2 whereas that seen with a
conventional microscopy is 0.02mm.2 Fluorescent microscopy requires much shorter time. A
microscopist can properly examine at least 100 smears per day by fluorescent microscopy
compared with only 30-40 ZN stained smears. Nevertheless in central or other large laboratories
where the work load exceeds that of 3 technicians working with 3 conventional microscopes
(more than 100-150 slides/day)6 it may be cheaper to use one fluorescence microscope instead.
Fluorescent staining 12 can detect bacilli when they are present around 10,000 bacilli/ml of
sputum, while Ziehl Neelsen staining requires 100,000 bacilli/ml to be detected in sputum India is
one of the high Tuberculosis burden countries in the world. In 2012 Auramine o staining based
Light emitting diode Fluorescence Microscopy (LED-FM) replaced conventional ZN microscopy in
200 designated microscopy centers of medical colleges operating in collaboration with RNTCP.
Our study compared both these methods. This Study was conducted at Department of
pulmonology in Katuri Medical College and Hospital, Guntur, Andhra Pradesh from 2012 to 2014.
AIMS: To compare the sputum smear examination by both fluorescent staining and ZN staining
methods in diagnosis of Pulmonary Tuberculosis and look for their cost effectiveness.
OBJECTIVES:
1. To check the percentage positivity, percentage negativity, acceptability of both these
methods.
2. To know the cost effectiveness and efficacy of fluorescent microscope for implementing the
same in general and peripheral institutions.
MATERIAL AND METHODS: In this study sputum samples were obtained from 200 patients.
Two sputum samples were obtained from each patient. One was spot sputum and the other was
early morning sputum, collected according to the RNTCP guidelines. From each sputum samples
two smears were prepared. In one smear, ZN staining was performed and in the other AR
staining was performed. They were compared with each other. So a total of 800 sputum smears
were examined from 200 patients.
INCLUSION CRITERIA:
1. Patients suffering from cough with expectoration for 2 weeks or more and with other
symptoms like shortness of breath, chest pain, evening rise of temperature, weight loss and
loss of appetite attending Katuri Medical College, Pulmonology OPD.
2. Patients of age greater than 18 years of age.
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EXCLUSION CRITERIA:
1. Patients who are taking ATT.
2. Patients less than 18 years of age.
3. Patients with active haemoptysis.
4. Insufficient samples, which means samples containing sputum less than 2ml.
5. Samples containing only saliva.
Total study population is 200 patients. The results of 200 patients after performing ZN
staining and AR staining have been analyzed.
The clinical data as per the proforma was reviewed for all patients and the following
observations were documented.
GENDER DISTRIBUTION OF PATIENTS: The study population consists of 130 males and 70
females. Males are more than females in this study population.
GRADING BY ZN STAINING:
Grading by Auramini-Rhodamine Staining:
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The mean age of the study population was 28.57. The predominant age group involved
were 19-28 years group and 59-68 years group followed by 49-58 years group. The least involved
group was 79-88 years age group.
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ORIGINAL ARTICLE
Difference in Grading between Auramine Rhodamine and Ziehl Neelsen method of staining.
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Out of 58 sputum positive patients, only 14 patients showed agreement with the grading
in both AR and ZN staining. These 14 patients were AR 3+ and ZN 3+. Statistical analysis of the
data was done by using Graph pad prism 5.0 version. P value is 0.0364(<0.05), statistically
significant. Chi-square Test value 4.379.
EVALUATION OF SYMPTOMS: Majority of patients presented with cough (100%) with or
without expectoration. The second most common symptom was SOB (34%), followed by fever
(21%). Other symptoms were weight loss (16.5%) and Haemoptysis (8%)
Out of the 58 patients positive for AFB right side was involved in 25 patients, left side
in 19 patients, bilateral in 12 patients and in two patients X-ray chest was normal:
Diabetes Mellitus type II is the most commonly associated co morbid condition in this Study seen
in 11% of individuals. Other co morbidities are Hypertension 10% of individuals, Coronary artery
disease in 5.5% of individuals and COPD in 10% of individuals. Depression and anxiety were
associated in Tuberculosis patients in a majority in our study.
COMPARISON BETWEEN THE SPOT SPUTUM SMEAR AND EARLY MORNING SPUTUM
SMEAR: Number of patients with spot sputum smears negative and early morning sputum
smears positive or scanty were 13(22.4%). Difference in Grading between spot sputum smear
and early morning sputum smear occurred in 27 patients (46.5%) of which early morning sample
is of higher grade than spot sputum sample.
TUBERCULOSIS WITH HIV INFECTION: In this study number of HIV positive patients were
10. Out of which 5 patients were sputum smear positive and 5 patients are sputum smear
negative.
Out of 142 sputum smear negative patients, 3 patients post bronchoscopy sputum was
positive for AFB.
Comparison of sex ratio in different studies: Our study showed male: female ration of
Tuberculosis patients of 2.62:1. This is probably because of more male patients seeking health
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ORIGINAL ARTICLE
care and probably under reporting. The table shows different studies with references showing
their ratios. Fear of enacted social stigma was the main concern for compliance with treatment
among women, who described they felt isolated and people no longer visited them or wanted to
have tea with them. (Johansson et al 1999). 56, 61 Bhanvalikar et al13 female 21%. Rajasekharan
et al found 76.3% incidence in male and 23.7% in female. K. Prashanthi et al14 found that 70%
of TB patients were males and 30% females. The increased occurence in males may be because
of increased attendance of malepatients to OPD(2,3,4)
Comparative analysis of different studies in comparison of ZN stain and Fluorescent
microscopy in different studies shown in the table
Sl.
No
1.
2.
3.
4.
5.
6.
Name of Study
K.Desai et al.
(12)(2006-2007)
Roman Goyal et al
(2011) (ref 4)
Balakrishna et
al.(Ref 18)
Prashanthi K et al
(ref 14)
Munshi SK et al
(Ref 74)
Githui et al
(Ref 53)
Percentage ZN
positive
Percentage Fluresescent
Mocroscopy positive
Difference in
percentage
19.88%(35/176)
38.63%(68/176)
18.75
7.47%(29/388)
14.69%(57/388)
7.22%
18.5%(37/200)
23.5%(47/200
05%
50%
69%
19%
12.56%
16.41%
3.85%
65%
80%
15%
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7.
8.
9.
10
Singh et al
(ref 75)
Ulukanligil et al
(Ref 16)
Heidi Slbert Study
(Ref 76)
Current Study
37.5%
42.5%
05%
67.6%
85.2%
17.6%
11%(39/355)
22%(78/355)
11%
20%(40/200)
29%(58/200)
9%
Cross sectional studies from Russia and Mecadonia showed the superiority of Fluorescence
microcscopy over conventional ZN microscopy. In Mecadonia sensitivity of the Lumin (LED
attachment on light microscope) and CFM were 87.8%, and that of restained ZN smears with
conventional light microscope was 78.0%. In Russia, sensitivity was as follows: Lumin 72.8%,
CFM 52.5%; re-stained ZN smears 28.5% and directly ZN stained smears 55.6%.(5)
Similar views were echoed by Ben J.marais et al(6) who observed that LED fluorescence
microscopy provides a reliable alternative to conventional methods and has many favorable
attributes that facilitate improved, decentralized, diagnostic services.
Laifangham’s et al17 in 2009 found that AR was found to be superior to ZN on several
aspects. The difference in their case detection rates was statistically significant (chi 2=24.93,
p<0.001). There was more agreement between culture and fluorescent microscopy 95.1% than
with ZN microscopy 69.6%. The percentage of false negative patients by AR staining was only
2.78% which was in sharp contrast to that of ZN (40.27%). AR was able to detect more
paucibacillary cases than ZN method.(7)
DISCUSSION:
1. India is a high burden country for Tuberculosis. TB is an Ubiquitous disease. In India.
Tuberculosis services are provided both in Public and Private Health Centers. TB is
diagnosed and treated at multiple levels.
2. Our study suggests that LED Fluorescent Microscopy is superior to conventional light
microscopy in the diagnosis of Pulmonary Tuberculosis. Very few Government laboratories
perform Fluorescent Microscopy. In Andhra Pradesh, only teaching hospitals (Tertiary care
Hospitals) are performing AR staining (Fluorescent Microscopy) under RNTCP. DTCs are not
equipped with Fluorescent Microscopes. AR stain is cheaper (69.9 paise per slide) than ZN
stain (72 paise per slide). The cost of Fluorescent Microscope is 4-5 times more than the
cost of conventional Light Microscope. General Practitioners should refer the patients to LED
Fluorescent Microscopy centers for sputum smear examinations rather than to ZN
Microscopy centers.
3. We calculated the cost of performing the ZN stain and fluorescent microscopy basing on the
cost of the slides and stains. The costs are comparable and cheap both the procedures
costing around a rupee each excluding the cost of the microscope and the technician’s
training and wages
4. Since early morning sputum is rich in Tubercle bacilli, the Present study insists on early
morning sputum smear examination.(8)
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5. On average time required to read a fluorescent slide is 2 minutes, whereas time required to
read a ZN Slide is 20 minutes. So, Fluorescent microscopy is quick for screening purpose.
6. FM can diagnose more number of Pauci bacillary TB than ZN microscopy(7) so it helps in
early diagnosis of TB, reducing the complications of TB such as Post Tubercular
Bronchiectasis, fibrosis, haemoptysis, Aspergillosis, BPF, Broncholith, Cavernolith and scar
carcinoma.
7. In 2009 WHO recommended, LED Microscopy be phased as an alternative for ZN
Microscopy(9) Globally the switch to LED Microscopes has been gradual, it was reported to
have occurred in only 6% of Microscopy centers in 2013.
8. One of the High burden countries like South Africa is having 100% LED Fluorescent
Microscopy Services.(9) 2. The Percentage of Microscopy centers in China using LED
Microscopes increased from 2% in 2012 to 33% in 2013 with Global Fund Support (WHO
report). Our neighbouring country Bangladesh is also having 17% (WHO report) of LED
Microscopy centers. India is lagging behind with only 2% of LED FMcenters (WHO data).
We should also increase our LED Microscopy centers in a huge way at community level, like
that of China with domestic and global funding support.
9. In the era of high degree of National and International travel, high level of education,
growing wealth of people and wide awareness, it is more apt for the people to have
accurate and earlier diagnosis of Tuberculosis. FM is suggested as more sensitive than ZN
microscopy by WHO also. Fluorescent staining is slightly cheaper than ZN staining. As LED
FM is more sensitive compared to ZN Microscopy, symptomatic sputum negative patients by
ZN microscopy should be subjected to LED microscopy, so that pauci bacillary cases can be
diagnosed early. This is more relevant for HIV TB patients as the disease is predominantly
pauci bacillary in HIV-TB co infection
So, we recommend to use LED Fluorescent Microscopy2 in a huge way in India at
peripheral Health care centers. In resource poor country like India it is achievable as 64%
of finances come from the Government and 36% finances from international funds.2
10. Further, the sensitivity of Fluorescence microscope is superior to ZN microscopy with only
very few false negatives in comparison(7) and the ability to detect pauci-bacillary cases
Fluorescence LED microscopy should be preferred in the RNTCP settings and the services
should be accessable to both Government and Private medical practitioners.
Diabetes mellitus, Hypertension, Coronary heart disease and HIV disease were associated
comorbid diseases in some of the TB paatients,. Depression and anxiety were seen in a majority.
Similar observation on depression and anxiety were seen by Aydin et al.(10)
SUMMARY:
1. Total study population consists of 200 patients. The results of 200 patients after performing
ZN staining and AR staining have been analyzed.
2. The study population consists of 130 males and 70 females. Males were more than females
in this study population.
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3. The study group varied in age between 19-88 years of which 130 were Males and 70 were
Females. The youngest patient was aged 19years and the oldest 88 years.
4. The mean age of the study population was 28.57.The predominant age group involved
were 19-28years group and 59 to 68 years group followed by 49 to 58 years group. The
least involved group were 79-88 years age group.
5. Out of 58 sputum smear positive patients,42 patients were males, 16 patients were females
with a male to female ratio of 2.62:1
6. The findings showed that Fluorescent staining and ZN staining preparation each showed
positive for AFB in 58(29%) out of 200 and 40(20%) out of 200 patients respectively. The
FM showed more sputum smear positivity than ZN Microscopy. 18 patients were positive by
AR Staining alone and negative by ZN staining.
7. Out of the 58 sputum positive patients, right side was involved in 25 patients chest x-rays,
left side in 19 patients chest x-rays, bilateral in 12 patients chest x-rays and normal in 2
patients. Right side was predominantly involved than Left side.
8. Number of patients with spot sputum smears negative and early morning sputum smears
positive or scanty are 13(22.4%).
9. Difference in Grading between spot sputum smear and early morning sputum smear
occurred in 27 patients (46.5%) of which early morning sample is of higher grade than spot
sputum sample.
10. Number of HIV positive patients were 10. Out of which 5 patients were sputum smear
positive and 5 patients were sputum smear negative.
REFERENCES:
1. Ravindran P, Joshi M, Sudheendra C and Janardhanan N.A Comparative Study on the
diagnostic efficacy and cost benefit of repeated sputum examination by Ziehl Neelsen
method, Fluorescent microscopy and culture in fresh clinically diagnosed cases of
Pulmonary Tuberculosis. Lung India Journal 1984. P117-201: 59.
2. Bhanvaliker J.N., Gupta R, Sharma DC, Goel MK.HIV seropositivity in hospitalized pulmonary
Tuberculosis patient in Delhi. Indian J Tubercle 1997: 44: 17-9.
3. K Prasanthi, AR Kumari, Efficacy of fluorochrome stain in the diagnosis of pulmonary
tuberculosis co-infected with HIV. Indian Journal of Medical Microbiology, 2005:23:179-185.
4. K. G. Purusotham et al: Comparative efficacies of three acid fast staining techniques under
field conditions for Mycobacterium Tuberculosis in the Indian context. Internet Journal of
Microbiology.2010 volume 9, NO. 1 accessed from
URL:http//www.ispub.com/journal/the_internet_journal_of_microbiology/volume9.
5. Comparison of Lumin™ LED fluorescent attachment, fluorescent microscopy and ZiehlNeelsen for AFB diagnosis-Authors: Trusov, A.1; Bumgarner, R.2; Valijev, R. 3; Chestnova,
R.3; Talevski, S.4; Vragoterova, C.4; Neeley, E. S.5Source: The International Journal of
Tuberculosis and Lung Disease, Volume 13, Number 7, July 2009,pp. 836-841(6) Publisher:
International Union Against Tuberculosis and Lung Disease.
6. Oxford Journals Medicine & Health Clinical Infectious Diseases Volume 47, Issue 2 Pp. 203207.
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7.
Laifangbam S et al: A comparative study of fluorescent microscopy with Ziehl Neelsen
staining and culture for the diagnosis Pulmonary Tuberculosis. Kathmandu Univ Med
Journal, 2009 Jul-Sep, 7(27) 226-30.
8. Ethiop Med J. 2012 Oct; 50(4): 349-54 improved detection of acid-fast bacilli in sputum by
the bleach-concentration techinique at Gondar University Teaching Hospital, northwest
Ethiopia.
9. WHO Global Tuberculosis Report 2014, Geneva, Switzerland. P 1-171. accessed from
URL:http://www.TB_REPORT_2014.pdf
10. Aydin IO, Ulusahin A, Depression, anxiety comorbidity, and disability in tuberculosis and
chronic obstructive pulmonary disease patients: applicability of GHQ-12. Gen Hosp
Psychiatry. 2001 Mar-Apr; 23(2):77-83.
LIST OF ABBREVIATIONS:
AFB
Acid Fast Bacilli
AIDS
Acquired immune deficiency Syndrome
AR
Auramine o rhodamine stain
ATT
Anti Tuberculosis Therapy
ART
Anti Retroviral Therapy
DOTS
Directly observed Treatment short course Therapy
HIV
Human immunodeficiency virus
M:F
Male:Female
MDR TB Multi Drug Resistant Tuberculosis
XDR TB Extensively Drug Resistant Tuberculosis
RNTCP
Revised National Tuberculosis control Programme
TB
Tuberculosis
WHO
World Health organization
ZN
Ziehl Neelsen staining
PCR
Polymerase Chain Reaction
iLED
Infrared Light Emitting Diode
LED
Light Emitting Diode
NO.
Number
SOB
Shortness of Breath.
FM
Fluorescent Microscopy
MPW
Multipurpose worker
DTC
District Tuberculosis center
QBC
Quantitative Buffy coat
DM
Diabetes Mellitus
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AUTHORS:
1. R. Ramakrishna
2. Chundari Bharavi
PARTICULARS OF CONTRIBUTORS:
1. Professor & HOD, Department of
Pulmonology, Katuri Medical College,
Guntur.
2. Post Graduate, Department of
Pulmonology, Katuri Medical College,
Guntur.
NAME ADDRESS EMAIL ID OF THE
CORRESPONDING AUTHOR:
Dr. R. Ramakrishna,
# A2, Raghavas Vista,
Krishna Nagar Park Road,
Guntur.
E-mail: ramakrishna45@yahoo.co.in
Date
Date
Date
Date
of
of
of
of
Submission: 01/02/2015.
Peer Review: 02/02/2015.
Acceptance: 28/02/2015.
Publishing: 16/03/2015.
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