Ziehl Neelsen stain sensitivity

In determining the etiologic diagnosis of patient’s tissue biopsy specimens

with granulomatous inflammation

Author: Wilda Mahdani1

Co-author: Ni Made Mertaniasih2, Arthur Pohan Kawilarang2, Troef
Soemarno3
1. Microbiology Department, Medical Faculty of Syiah Kuala University,
Darussalam, Banda Aceh 23111. Phone: (0651)7551843, email:
[email protected]
2. Microbiology Department, Medical Faculty of Airlangga University,
Surabaya.
3. Pathology Department, Medical Faculty of Airlangga University, Surabaya.

Abstract
The main infectious agent cause granulomatous inflammation is
Mycobacterium tuberculosis. There are a lot of forms and patterns of tuberculosis
infection. Clinicians are generally more suspicious of pulmonary infection, while
infection of tuberculosis in the other tissue is often overlooked. This observational
descriptive study aimed to find out the sensitivity of Ziehl Neelsen (ZN) stain in
determining etiologic diagnosis of granulomatous inflammation on
histopathological specimens. Study samples were 37 pieces tissue blocks which
diagnosed as granulomatous inflammation.
The study found positive Acid Fast Bacilli (AFB) in 23 tissue block.The
conclusion of this study was ZN staining technique had sensitivity 81%, specificity
90%, Positive Predictive Value (PPV) 96% and Negative Predictive Value (NPV)
64% to determine the etiologic diagnosis of specific granuloma. The incidence of
mycobacteria infection causes granulomatous inflammation in the sample studied
was 62.16%. Special staining techniques can be used to diagnose the etiology of
tissue infection that will direct patients to appropriate therapy.
Keywords : Granulomatous inflammation, Specific granuloma, Ziehl-Neelsen
Staining

Introduction
Publications about the histopathological evaluation of tissue specimens in
infectious diseases management are still uncommon.1 One of the important roles
of microbiology diagnostic laboratory is tissue examination, while cytopathology
experts usually the first one to evaluate tissue lesions case both infectious and
non- infectious.2 Good cooperation between the two divisions will bring great
benefits to many parties.
Tuberculosis has caused high morbidity and mortality throughout the history of
mankind .3 Tuberculosis remains a major health problem in the world today,

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especially in developing countries. Diagnosis of tuberculosis is determined by the
discovery of acid fast bacilli in smear and culture of mycobacteria. Early diagnosis
facilitate for specific antimycobacterial therapy. Tuberculosis also can be
diagnosed as a chronic granulomatous inflammation histopathologically.4
Diagnosis of tuberculosis is still a challenge, with regard to many differential
diagnosis. Histopathological picture of granulomatous inflammation can also be
found in various states in addition to tuberculosis, such as foreign body reactions,
fungal infections, sarcoidosis, cat scratch disease, leprosy and brucellosis.4
Medical history and physical examination is essential. Granuloma is a chronic
inflammatory focus consisting of microscopic aggregation of macrophages
transformed into cells resembling epithelium, surrounded by a number of
mononuclear leukocytes, principally lymphocytes and occasionally plasma cells.5
Each of diagnostic tests has advantages and limitations. Tests that are often
performed include sputum smear, culture, fine needle aspiration cytology (FNAC),
tissue biopsy and polymerase chain reaction technique (PCR).6
The benefits of a diagnostic examination is its ability to detect a person
suffering from the disease and determine someone without the disease. These
capabilities are described by using several measures, namely sensitivity,
specificity, positive predictive value (PPV), and negative predictive value (NPV).
Sensitivity is the proportion of people who actually suffer from the disease with a
positive test result. Specificity is the proportion of people who do not have a
disease with a negative test result. Positive predictive value (PPV) is the
proportion of people who have a positive test result who actually have disease,
while the negative predictive value (NPV) is the proportion of people who have a
negative test result that really have no disease.7
This research aims to study the sensitivity of ZN staining to determine the
etiologic diagnosis of inflammatory granulomatous tissue specimens, the
distribution and frequency of infection and the distribution and frequency of
mycobacteria infection by age, sex, and tissue origin.

Materials and Methods

This study was an observational descriptive. Patients data taken from medical
documents of Pathology department Dr. Soetomo Hospital, Surabaya. Paraffin
embedded tissue blocks with granulomatous inflammation then sought and
2
collected. The research samples are blocks with pathologic diagnosis specific and
nonspecific granulomas, obtained 37 blocks. Examined tissue taken from various
organs; such as the lungs, lymph nodes, skin, bones, and so on.
1. Object glass processing with AES (Aminopropyltriethoxy silane) reagent.
Object glass processed by AES reagent to immobilized tissue sliced and do not
shed in ZN staining. AES 2% solution (245 ml acetone, 5 ml
Aminopropyltriethoxy silanes) prepared, distilled water I and II in the staining
jar. Slide laid out in a rack, put in a solution of AES for 2,5 minutes. Slide rinsed
in distilled water I dipped by as much as ten times, and then proceed to the
distilled water II, rinse as much as ten times. Slide dried, slides arranged in a
grid. Slide put in an incubator with a temperature of 37 ° C for 24 hours.
2. Tissue sectioning
Tissue blocks leveled surface (trimming), then made 3-5 μm slices. Tissue
sections put in 41-42oC water bath for one minute. Pieces are then glued to a
slide that has been processed with AES, then dried.
3. Deparaffinization
This process aims to eliminate the paraffin in tissue section prior to staining.
Slides dipped in xylene I for two minutes, then transferred to xylene II for two
minutes. Slides then transferred to absolute alcohol for 30 seconds and I
absolute alcohol II for 30 seconds. The final stage slides put in 70% alcohol for
30 seconds and rinsed with distilled water.
4. Ziehl Neelsen Staining
This staining technique relies on the acid resistance of germs. Tissue sections
put into distilled water. Carbol fuchsin is poured into the top of the slide by first
putting a square piece of filter paper on top of tissue sections to prevent the
deposition of dyes. Slide warmed until steamy: this can be easily done by using
the flame from throat swab dipped in rubbing alcohol. Heating is carried out for
15 minutes. Slide then rinsed with distilled water and then given acid alcohol
until pale pink tissue and no more color wear off (about 1-3 minutes). Repeated
rinsing with distilled water. Giving counterstain with methylene blue acid for
three minutes. Slide then dried and dehidrated with xylene twice, the length of
each was five minutes. The final stage was mounting with synthetic resin
medium. Positive and negative control slides are always used when staining.

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 The results will show a red acid fast bacteria, other bacteria are blue, cells and
cell nuclei in blue, and erythrocytes will be slightly red.8
5. Data analysis
Patients age, sex, tissue, positive cases are presented in the form of
distribution and frequency. Analysis of data to calculate sensitivity and
specificity by using a 2x2 table or by using Epicalc 2000 software.
Result
The youngest patients with granulomatous inflammation in this study was
two months old and the oldest was 74 years old, the average age was 28.11
years. Women patients were 21 and men were 16.

Table 1. Patients sex and age.

Sex Number Age
Male 16 2 month – 52 year
Female 21 2 year – 74 year
TOTAL 37

Clinical diagnosis is made by clinical examination based on medical history

and physical examination before specimens sent to the laboratory. The clinical
diagnosis of tuberculosis found in 16 people (43.24%) patients and 21 patients
were diagnosed with non-tuberculosis.
12

10

6 Male
Female
4

0
Tuberculosis Non-Tuberculosis

Picture 1. Clinical diagnosis distribution according to patient’s gender.

Distribution of clinical diagnosis in different tissues were as follows:

4
 8
7
6
5
4
3 Tuberculosis
2
Non Tuberculosis
1
0

Picture 2. Distribution of clinical diagnosis in different tissue specimens.

The clinical diagnosis of tuberculosis most found in bone specimens which
were all from the vertebrae, all clinical diagnosed with spondylitis tuberculosis.
Lymph nodes specimens which diagnosed lymphadenitis tuberculosis found in 3
blocks, the rest were diagnosed lymphadenopathy. Other clinical diagnosis of
tuberculosis was found in skin specimens as much as two blocks, two blocks lung
specimens, one cerebellum block, as well as the synovium, larynx and omentum
of each single block.
ZN staining results on samples with clinical diagnosis of TB and non-TB is
as follows:
14
12
10
8
Tuberculosis
6
Non Tuberculosis
4
2
0
ZN + ZN -

Picture 3. ZN staining results on the distribution of the sample based on clinical

diagnosis.

ZN staining results showed besides positive AFB in 13 samples with diagnosis

of tuberculosis, were also found in 10 samples with diagnosis non-Tuberculosis.
This suggests that tuberculosis tissue lesions were difficult to be determined
clinically.

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 Pathologic diagnosis of tissue specimens were established based on the
assessment with Hematoxilin Eosin (HE) staining tissue reaction, it showed a
picture of specific granulomas for tuberculosis diagnosis and non-specific
granulomas for other than tuberculosis. Classic characteristics of tuberculosis
granulomas is the central caseating necrosis known as tubercles. This area is
covered by epithelioid cells, lymphocytes, histiocytes, fibroblasts, and occasional
Langhans giant cells.9
16
14
12
10
8 Male
6 Female
4
2
0
Specific granuloma Nonspecific granuloma

Picture 4. Distribution of pathological diagnosis proportions by sex of patients.

Pathologic diagnosis was made based on the assessment of tissue
reactions with HE staining showed 27 specific granuloma specimens and ten
nonspecific granuloma specimens. Specific granulomas were obtained from 12
male patients and 15 female patients. Nonspecific granulomas were obtained
from four male patients and six female patients.
Picture of specific granuloma found in 27 (72.97%) specimens. All lymph
node specimens, bone and intracranial tissues revealed specific granuloma.
Other specific granuloma pattern found in seven specimens of lung, four skin
specimens, one nasal mucosa tissue specimen, one palate specimen and one
omentum specimen.

6
 8
7
6
5
4
3 Specific granuloma
2
Nonspecific granuloma
1
0

Picture 5. Distribution of pathologic diagnosis in various tissue specimens.

ZN staining of the entire tissue sections was conducted to identify the

presence of AFB in the tissue specimens. This examination determined etiologic
diagnosis of granulomatous inflammation caused by mycobacteria infection.
Identification of mycobacteria species can not be done with the staining technique,
but should be by culture or molecular techniques ..
8
7
6
5
4
3 ZN +
2
ZN -
1
0

Picture 6. Distribution of ZN staining results in tissue specimens

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Picture 7. ZN staining results in lung tissue showed a positive AFB. Showing red
bacilli among the cells of the lung parenchyma (1000x magnification).

Picture 8. ZN staining results on vertebrae specimen showed a positive section.

Showing the collection of red acid fast bacilli among the osteocytes and the
necrosis bone tissue (1000x magnification).

Picture 9. ZN staining results in cerebellar tissue showed positive AFB. Showing

the collection of AFB among cerebellar parenchymal cells (1000x magnification).

Most positive result were found in lung specimens, the lymph nodes, and
bones. Skin specimens showed only two positive samples. Seven lung specimens
showed positive results. Other positive results observed in the two skin
specimens, five lymph nodes specimens, five bone specimens, two nasal mucosa
specimens, and two intracranial specimens.

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 25

20

15
Specific granuloma
10 Nonspecific granuloma

0
ZN + ZN -

Picture 10. Distribution of ZN staining results for pathological diagnosis.

Positive AFB observed in 22 (81.48%) of 27 specimens that have specific

granuloma patterns. One sample with a diagnosis of nonspecific granulomas also
showed a positive result, i.e. nasal mucosa tissue biopsy specimens.

Table 2. Cross tabulation of ZN staining results with pathological diagnosis.

Specific Nonspecific
Total
granulomas granulomas
ZN + 22 1 23
ZN - 5 9 14
Total 27 10 37

Sensitivity of ZN staining technique to determine the etiologic diagnosis of a

specific granuloma is 81%. This means that the proportion of patients with
specific granulomas who showed positive ZN results was 81%. Specificity was
90%, which means that 90% of people who do not have specific granuloma will
show the negative results of the examination.
The proportion of people that show positive test results are really had
specific granulomas was 96%, in other words, this examination has Positive
predictive value (PPV) of 96%. Negative predictive value (NPV) of the
examination was 64% which means that the proportion of people that show a
negative test result that really did not have specific granuloma is 64%.

Discussion
The youngest patient in this study was two months old and the oldest was
74 years old with an average age of 28.11 years. Female patients (21 persons)

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more than men (16 persons). These data supported previous theory which states
that tissue infections can occur at any age and without any predisposing factors,
although immunosuppression and drug abuse increases the risk.10
The results showed a positive AFB in 23 tissue specimens (62.16%). Most
positive ones were found in lung specimens, the lymph nodes, and bones. Skin
specimens showed only two positive samples. Positive smear was also found in
22 (81.48%) of 27 specimens that have specific granuloma patterns.
Tuberculosis can be spread widely, rarely organ or tissue that can not be
attacked by tuberculosis. The most commonly involved organs was lungs.6
Infection by M. tuberculosis and other mycobacteria can occur in almost every
organ. Lymphadenopathy is the most common, but other sites like oropharynx,
mammary, thyroid, skrotal lesions, para-spinal lumps, bone lesions, parietal
viscera of the anterior abdominal wall, skin and subcutaneous, intra-abdominal
lesions, tubo-ovarian pelvic masses had been reported.2
Sensitivity of ZN staining technique to determine the etiologic diagnosis of a
specific granuloma is 81%. This means that the proportion of patients with
specific granulomas who showed positive ZN results was 81%. Specificity was
90%, which means that 90% of people who do not have specific granuloma will
show the negative results of the examination. The proportion of people that
show positive test results are really had specific granulomas was 96%, in other
words, this examination has Positive predictive value (PPV) of 96%. Negative
predictive value (NPV) of the examination was 64% which means that the
proportion of people that show a negative test result that really did not have
specific granuloma is 64%.
Ziehl Neelsen staining sensitivity is not as high as its specificity, in
accordance with previous studies. This can happen due to too small number of
bacteria, and the results can not distinguish M. tuberculosis from other
Mycobacterium sp.11
Karuniawati et al. in a study about microscopic examination of sputum
stated that microscopic examination of sputum with ZN staining is the simple, fast,
inexpensive, and sensitive enough to support the diagnosis of tuberculosis and to
assess the progress of treatment. Sensitivity of Ziehl Neelsen obtained for 81.5%,
and specificity 91.6. Positive predictive value was 78.6%, while the negative

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predictive value was 92.9%. The study concluded that the Ziehl Neelsen
technique is the best method and can be performed in a simple laboratory.11

Conclusion
ZN staining technique had sensitivity 81%, specificity 90%, Positive Predictive
Value (PPV) 96% and Negative Predictive Value (NPV) 64% to determine the
etiologic diagnosis of specific granuloma. The incidence of mycobacteria infection
causes granulomatous inflammation in the sample studied was 62.16%.
History of the disease and an assessment of macroscopic and microscopic
tissue biopsy specimens is very important to find out the microorganisms that
caused tissue infections. Special staining techniques can be used to diagnose the
etiology of tissue infection that will direct patients to appropriate therapy.

Acknowledgment
Thanks to Dr. Budi Utomo, dr., M. Kes, Abu Rohiman, dr., MS, Sp.MK (K), and
Bambang Susilo, dr., M. Kes, Sp.MK (K) for their helpful knowledge. Thanks to the
staff of Microbiology and Pathology Department of Medical Faculty, Airlangga
University and Dr. Soetomo Hospital, for their help over the research days. Special
thanks to Christina Susilo for her priceous time in conducting this research.
Hopefully, this writing can benefit all of us, the author and co-authors humbly
apologize for any shortcomings.

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