COMPARISON OF AURAMINE-O STAIN AND ZIEHL-NEELSEN STAIN IN THE DETECTION OF TUBERCULOUS BACILLI IN FINE NEEDLE ASPIRATION OF LYMPH NODES.
Tuberculosis (TB) is one of the major public health problems, as it is an easily communicable disease, especially in developing nations like India. According to a recent statistics, the incidence in India is about 2.2 million cases out of a global incidence of 9.6 million tuberculosis cases.  The two types of clinical manifestations of TB are pulmonary and extrapulmonary tuberculosis (EPTB). EPTB is defined according to WHO classification criteria as, "An infection by M. tuberculosis which affects the tissues and organs outside the pulmonary parenchyma" and it represents 20-25% of all the TB cases.  A patient with both pulmonary and extrapulmonary tuberculosis is classified as a case of pulmonary tuberculosis. Burden of EPTB is high and ranges from 15-20% of all TB cases in HIV negative patients and accounts for 40-50% in HIV positive patients. 
The most common presentation of EPTB is peripheral lymphadenopathy. [4, 5] However, diagnosing EPTB remains challenging because clinical samples obtained are paucibacillary, decreasing the sensitivity of diagnostic tests. Fine Needle Aspiration Cytology (FNAC) is now widely utilized as a first line diagnostic procedure in the diagnosis of peripheral lymphadenopathy and its role is well documented. 
The diagnosis of TB adenitis can be made by utilizing many different techniques, including cytomorphology, special stains to identify the organism, culture and Polymerase Chain Reaction (PCR). Among these, culture (gold standard) is essential for obtaining a definitive diagnosis but is time-consuming and requires specialized safety procedures in laboratories. PCR, although rapid, is too costly to be routinely used. 
For cytopathological diagnosis, presence of epithelioid cell clusters with caseous necrosis has been commonly used to define a positive smear. However, loss of host immune function (conditions like AIDS) can result in greater suppurative response and less well-formed epithelioid cell granulomas. Additionally, the epithelioid cell granulomas are also seen in non-tuberculous mycobacterial disease, fungal infections, brucellosis or syphilis, so cautious interpretation is required. Among the special stains used, the conventional Ziehl-Neelsen (ZN) method for acid-fast bacilli (AFB) plays a key role in the diagnosis and also in monitoring the treatment but has low sensitivity ranging from 20%-43%. [8, 9]
A method for identification of AFB which is more sensitive than conventional ZN method is required for early diagnosis of tuberculous lymphadenitis. Auramine-O staining technique plays a significant role in the detection of Mycobacteria in the present days. The main aim of this study is to compare the efficiency between Auramine-O staining method and Ziehl-Neelsen method in the detection of tuberculous bacilli and to prove the diagnostic value of Auramine-O stain in hospitals where fluorescent microscopy is available, along with routine H&E stain or Papanicolaou stained lymph node aspirates.
MATERIALS AND METHODS
This analytical cross-sectional study was conducted in the FNAC clinic, Department of Pathology on 50 patients from July 2017--September 2017. All patients presenting with peripheral lymphadenopathy with high clinical suspicion of tuberculous aetiology were included in the study. Patients with primary malignancy and lymphadenopathy with suspicion of secondary deposits and inadequate aspiration smears were excluded from the study.
After obtaining the consent, fine needle aspiration was done using standard protocol and a minimum of three smears were prepared from each of the aspirates. One smear was alcohol fixed for Haematoxylin and Eosin (H&E) staining and the other two were air dried for Ziehl-Neelsen and AuramineO staining respectively. Relevant investigation details such as haematology, chest radiogram was reviewed in these patients.
Ziehl-Neelsen Staining Procedure
The air-dried smears are gently heat fixed and are stained with strong carbol fuchsin and intermittently heated for 5-10 minutes. The slide is washed with tap water and decolourised with acid alcohol (20% sulphuric acid with 95% ethanol) for two minutes. After washing the slide with tap water, counter staining is performed with Gabbots methylene blue for 5-10 minutes. The slide is washed with tap water and examined under oil immersion objective (100x). The acid -fast bacilli was seen as bright pink curve or straight rods against a blue background.
Auramine-O Staining Procedure
The smears are air dried for 5 minutes and heat fixed for 2-3 minutes. The heat fixed smears are stained with 0.1% Auramine-O stain for 20 minutes. The slide is washed under tap water and decolourised with acid alcohol (0.5% hydrochloric acid with 70% ethanol) for 2-4 minutes. After washing the slide with tap water, counter staining is performed with 0.5% potassium permanganate for 30 seconds. The slide is washed with tap water, air dried and examined under high power objective (40x). Tuberculous bacilli appeared as bright brilliant yellow rods against a dark green background.
Haematoxylin and Eosin Staining Procedure
Fix the smears for 15 minutes in alcohol. Add haematoxylin to the smear and wait for 15 minutes. After washing it with tap water, allow it for blueing under running tap water for 10 to 15 minutes. Stain it with eosin by making 2 to 3 dips. Wash it under tap water. Mount it with DPX mountant and visualize under microscope.
The data obtained were processed using chi-square test and the results showed statistical significance. The chi-square value is 5.8644 and the p value is 0.0154 (significant association at p<0.05).
A total of 50 fine-needle aspiration specimens from lymph nodes were evaluated in this study. The age ranged from 2 to 70 years, with the mean age of 23.4 [+ or -] 13.5 years. Female preponderance was noted accounting for 64% (32/50) of cases. There were 13 HIV positive cases (26%) and 37 HIV negative cases (74%) [Figure 2]. Most commonly involved lymph nodes in our study were cervical (80%) followed by axillary (16%) and inguinal (4%) groups [Figure 3]. Of the 50 lymph node aspirates, the smear positivity for AFB on the conventional Ziehl-Neelsen method was 44% while the smear positivity increased to 80% on the Auramine-O staining method. The staining of the lymph node smears with Auramine-O increased the smear positivity by 36% over the Ziehl-Neelsen method. 19 cases which were negative by ZN method were diagnosed positive by Auramine-O staining method [Table 1]. Also, one specimen diagnosed as tuberculous lymphadenitis on ZN method, showed negative result by Auramine-O method which may be due to the decreased density of the bacilli.
Among the 50 lymph node aspirates, 54% (27/50) of the smears showed epithelioid cell granulomas in a background of caseous necrosis [Figure 4], which are characteristic of TB adenitis but 46% (23/50) of them showed only necrotic debris which was suggestive of suppurative lymphadenitis. The possible explanation is that in immunosuppressive conditions like AIDS, there is no sufficient formation of epithelioid cell granulomas, so that it is possible to miss the diagnosis [Figure 5]. Out of these 23 cases, AFB were demonstrated in 20 cases by Auramine-O method and in 13 cases by Ziehl-Neelsen method.
India has a long history of research and demonstration projects on tuberculosis.  The detection of acid-fast bacilli (AFB) is often considered as the evidence of the infected state. Thus, the laboratory plays a critical role in the diagnosis of TB.  In developing countries, microscopy of the specimen is by far the fastest, cheapest and the most reliable method for the detection of AFB. 
The use of Auramine-O stain is recommended because of its increased sensitivity and ease of interpretation compared with the ZN method.  The present study was attempted to use the Auramine-O stain and compare it with the conventional Ziehl-Neelsen stain on lymph node aspirates. The staining of the lymph node smears with Auramine-O increased the smear positivity by 36% over the conventional ZN method [Table 1]. The Auramine-O staining technique greatly improves the diagnostic value especially in patients with low bacillary load that are likely to be missed on Ziehl-Neelsen stained smears [Figure 7].
Under fluorescent microscope, AFB typically appear as golden yellow rods against a dark green background [Figure 6]. Although the ability to retain Auramine-O stain, after washing with alcohol or weak acids is a primary feature of the genus Mycobacterium, it is not entirely unique to the genus. Other bacteria, which contain mycolic acids, such as Nocardia, can also exhibit this feature. However, good observation is required to distinguish with certainty AFB from other small, naturally fluorescent particles present in some smears. The acid -fast bacilli were seen as bright pink curve or straight rods in Ziehl-Neelsen staining method. [Figure 8].
Previous studies on sputum smears have shown the positivity rates for both the Ziehl-Neelsen and Auramine-O staining methods as 65% and 80% respectively.  But, in our study on lymph node aspirates in cytology, the positivity rates were 44% for the ZN method and 80% for the Auramine-O staining method [Figure 1]. All these AFB positive patients responded well to the anti-tubercular therapy. The results of the present study are in concordance with the study conducted by Vamseedhar Annam et al. [Table 2]. The Auramine-O staining technique is more sensitive than conventional Ziehl-Neelsen staining method as even low bacillary load can be diagnosed easily under fluorescent microscope and it has been superior to the ZN staining. 
At present, Catridge Based Nucleic Acid Amplification and Testing (CB-NAAT) is used only for sputum testing in pulmonary tuberculosis. This can be extended to aspirates from extrapulmonary lesions as well, to increase the detection rate of tuberculous bacilli. Providing adequate training about FNAC of suspicious tubercular nodes and staining technique to health care professionals at all levels of health care system will improve detection of extra-pulmonary tuberculosis in early stage itself.
Auramine-O staining method was found to be more efficient and advantageous than conventional ZN staining method particularly in paucibacillary cases. Since Auramine-O stained smears are scanned under lower magnification (20x) than Ziehl-Neelsen stained smears (100x), a greater area is screened per field which makes the process less time-consuming and reduces observer fatigue. High initial cost of the equipment will always be a factor but where facilities are available, its use should be considered seriously. The use of Auramine-O stain alone could not be an alternative to conventional Ziehl-Neelsen staining. Hence, it would be beneficial if we use it as an adjuvant along with routine cytology for the early diagnosis of tuberculous lymphadenitis.
Auramine-O staining technique was found to be more efficient and advantageous than conventional ZN staining method especially in cases with low bacterial load. Since Auramine-O stained smears are scanned under lower magnification (20x) than Ziehl-Neelsen stained smears (100x), a greater area is screened per field which makes the process less time-consuming and reduces observer fatigue. High initial cost of the equipment will always be a factor but where facilities are available, its use should be considered seriously. The use of Auramine-O stain alone could not be an alternative to conventional Ziehl-Neelsen staining. Hence, it would be beneficial if we use it as an adjuvant along with routine cytology for the early diagnosis of tuberculous lymphadenitis.
This study was approved and funded by The Indian Council of Medical Research--Short Term Studentship 2017.
The Institutional Human Ethics Committee approval was obtained (No. 011/2017).
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R. S. Lokeshwaran (1), K. B. Lavanya (2), A. Dhanalakshmi (3), S. Keerthivasan (4), S. Yogalakshmi (5)
(1) 3rd Year MBBS, Coimbatore Government Medical College, Coimbatore, Tamilnadu, India.
(2) Assistant Professor, Department of Pathology, Coimbatore Government Medical College, Coimbatore, Tamilnadu, India.
(3) Associate Professor, Department of Pathology, Coimbatore Government Medical College, Coimbatore, Tamilnadu, India.
(4) Professor and HOD, Department of Thoracic Medicine, Coimbatore Government Medical College, Coimbatore, Tamilnadu, India.
(5) Senior Assistant Professor, Department of Pathology, Coimbatore Government Medical College, Coimbatore, Tamilnadu, India.
'Financial or Other Competing Interest': Dr. Lokeshwaran reports grants from The Indian Council of Medical Research--Short Term Studentship 2017, during the conduct of the study.
Submission 23-10-2018, Peer Review 17-11-2018, Acceptance 23-11-2018, Published 03-12-2018.
Dr. K. B. Lavanya, L-701, Purvabluemont, Near Shanthi Social Service, Trichy Road, Coimbatore-641004, Tamilnadu, India.
Caption: Figure 4. Haematoxylin and Eosin Stained Smear showing Epithelioid Cell Clusters in a Background of Neutrophils (40x)
Caption: Figure 5. Haematoxylin and Eosin Stained Smears showing Suppurative Lymphadenitis with Subtle Collection of Epithelioid Cell Clusters (10x)
Caption: Figure 6. Auramine-O Stained Smear Showing Abundant Golden, Slender, Rod-Shaped Tuberculous Bacilli (20x)
Caption: Figure 7. Auramine-O Stained Smear showing Scanty Tuberculous Bacilli (20x)
Caption: Figure 8. Ziehl-Neelsen Stained Smear showing Acid-Fast Bacilli (100x)
Table 1. Comparison of Smear Examination Result by Ziehl-Neelsen and Auramine-O Staining Techniques Ziehl-Neelsen Method Auramine-o Method Total Positive Negative Positive 21 01 22 Negative 19 09 28 Total 40 10 50 Table 2. Comparing the Smear Positivity Results of Present Study with Other Studies Smear Positivity by Smear Positivity Ziehl-Neelsen by Auramine-O Method Method Present study 44% 80% Vamseedhar et al 44.11% 81.37% A.Jain et al 22% 52% Laifangbam et al 25% 71.6% Habeenzu et al 21% 57% Figure 1. Correlation of the Diagnostic Accuracy among H&E, Ziehl-Neelsen and Auramine-O Staining Methods STAINING TECHNIQUES POSITIVE NEGATIVE H&E 54% 46% ZIEHL-NEELSEN 44% 56% AURAMINE-O 80% 20% Note: Table made from bar graph. Figure 2. A total of 13 Patients (26%) were HIV Positive and the Remaining 37 Patients (74%) were HIV Negative Prevalence of HIV POSITIVE 26% NEGATIVE 74% Note: Table made from pie chart. Figure 3. Among the 50 Lymph Nodes Studied, Aspirates were from Cervical (n=40), Axillary (n=8) and Inguinal (n=2) Groups Distribution of lymph nodes AXILLARY 16% INGUINAL 4% CERVICAL 80% Note: Table made from pie chart.
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|Title Annotation:||Original Research Article|
|Author:||Lokeshwaran, R.S.; Lavanya, K.B.; Dhanalakshmi, A.; Keerthivasan, S.; Yogalakshmi, S.|
|Publication:||Journal of Evolution of Medical and Dental Sciences|
|Date:||Dec 3, 2018|
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