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Background: Ziehl–Nelson (ZN) method for acid-fast bacilli (AFB) plays a key role in the diagnosis and monitoring of treatment in tuberculosis. Its major disadvantage is low sensitivity, ranging 9 to 46%. Mycobacterial culture is the reference method for detection of tubercle bacilli, but it is time consuming and requires specialized safety procedures in laboratories. Serological techniques have the disadvantage of lack of sensitivity and specificity. Newer molecular techniques, such as polymerase chain reaction (PCR), are costly to be routinely used in developing countries where tuberculosis is prevalent.
Microscopy has many advantages when it comes to speed and feasibility, and if its sensitivity could be improved, it has the potential to be a more valuable tool for tuberculosis control programmes around the world. Thus the need for new, improved, low-cost techniques cannot be over emphasized.
Aim : To evaluate improved sensitivity of bleach concentration method over conventional ZN stained smears on fine needle aspirates of lymph nodes in the diagnosis of tubercular lymphadenitis.
Materials and method:
198 clinically suspected cases of tuberculosis with enlarged peripheral lymph nodes attending the Department of Medicine, Surgery, ENT, TB and Chest medicine were subjected to FNAC. Smears were prepared and stained by ZN stain by conventional method as well as by treating with modified bleach method respectively and evaluated for acid fast bacilli.
The modified bleach method treatment of fine needle aspirates before ZN staining is able to detect more AFB as compared to conventional direct ZN stain. Bleach treated ZN smears had detected additional 62/150(41.66%) cases which were negative on direct ZN smears and increased the smear positivity by 41.33%.
Conclusion: This study suggests that the bleach method can significantly improve the diagnostic yield, 61.33% increased detection of AFB by microscopy for tuberculous lymphadenitis. Modified bleach method is safe, easy to perform, faster and inexpensive method to diagnose a tuberculous lesion and can be useful in routine cytology.
Tuberculosis is a great problem throughout the world, especially in developing countries like India. Beside pulmonary tuberculosis, extra pulmonary tuberculosis is relatively more common among the Asian population. Tuberculosis of the lymph node (tuberculous lymphadenitis) is the most common form of extrapulmonary tuberculosis.
In developed countries, tuberculosis is implicated in as few as 1.6% of patients with lymphadenopathy. But in developing countries almost two third of the cases of lymphadenopathy are due to tuberculosis. Tuberculosis should be strongly suspected in a young patient presenting with peripheral lymphadenopathy, with prolonged duration of illness, and involvement of cervical glands with multiple and matted appearance.
Early diagnosis is the corner stone of tuberculosis control strategies. Clinical diagnosis of tubercular lymphadenitis of the superficial lymph nodes is easy when features like matting, ulceration, sinuses, Caseation and liquefaction are present. In the early cases and some of the late cases these features are absent resulting in diagnostic difficulties.
The usual diagnostic methods employed are histopathology, smear microscopy, and mycobacterial culture on biopsy specimens. Diagnosis is often made on histopathology. Diagnosis is made when there is caseation necrosis in a granulomatous lymphadenitis. Ziehl Neelsen staining (Z. N. Staining) on the histopathologic section is an adjunct. However the chances of identifying acid fast bacilli in a tissue section are much lower. The effect of formalin and xylene on the stainabilty of mycobacteria by Ziehl Neelsen method results in extreme low sensitivity of detection of mycobacteria in histopathologic section.
We used the bleach concentration method on fine needle aspirates and compared the sensitivity of this technique in detecting AFB with that of routine ZN staining on aspirate smears. This study has been taken up to validate the studies done earlier.
MATERIALS AND METHODS
The study was conducted at the Department of Pathology (cytology laboratory) in a government medical college for a period of 1 years 5 months i.e., from June 2014 to October 2015.
198 clinically suspected cases of tuberculosis with enlarged peripheral lymph nodes attending the Department of Medicine, Surgery, ENT, TB and Chest medicine were subjected to FNAC. Out of 198 cases, 21 were diagnosed as reactive lymphadenopathy,19 were acute suppurative inflammation, 03 were metastatic lesions and 05 samples were inadequate for evaluation. Statistical analysis of 150 samples was evaluated.
Procedure of F.N.A.C and Bleach method FNA was performed with a 19-gauge needle and a 10-mL disposable syringe fitted with a syringe holder. Following lymph node aspiration, approximately 3-4 smears were made on clean glass slides. One or two smears were fixed in 100% methanol and stained with H&E stain. Air-dried smears were stained using the routine ZN method. The bleach method was performed with the remaining aspirated specimen in the syringe or needle hub, which was rinsed with 1 ml normal saline and transferred into 5 ml sterile disposable, conical screw-capped tubes. To this conical tube, 2 ml of 5% NaOCl was added and the mixture was incubated at room temperature for 15 min by shaking at regular intervals. The conical tube containing the mixture was concentrated by centrifugation at 3000 rotations per minute for 15 minutes after addition of 2 ml of distilled water. The supernatant was carefully discarded and the sediment was transferred with a sterile pipette on to a clean sterile slide. The slide was air-dried, heat fixed and stained by the ZN method. As a control, 2 ml of distilled water was centrifuged and the sediment was stained by ZN staining to rule out any error due to contamination while testing each specimen. The slides were mounted and screened for bacilli at oil immersion(1000x). Positive control-smears of sputum positive for AFB by ZN method were used.
The Sodium hypochlorite (NaOCl) solution: The household bleach was purchased, in which the stated chlorine concentration was 5%. To prevent the reduction of the chlorine activity due to a repeated exposure to air, each 5 L bottle was decanted after it was opened into a 25ml brown glass bottle for daily use and the remaining solution which remained unused at the end of the day was discarded. A ZN smear was made from each new bottle to ensure no contamination of mycobacteria.
Bleach method can result in fragile smears and can be washed off during slide staining, so care was taken to avoid this problem ,also overheating of the slides was avoided as it may result in the formation of crystals of hydroxide, which may put a compromise on the readings.
All the direct smears were read by one microscopist and the bleached smears were read by another microscopist and at the end of the study, the collection smears were swapped between the two microscopists. The microscopist who read the direct smear first, now read the bleached smear and the other microscopist who read the bleached smear first, now read the direct smear. The results were compared by one of the invesigators who examined the discordant slides and discussed with both the microscopists to reach a consensus.
Results were expressed using ATS (American Thoracic Society) scale.A negative report was not given till at least 100 fields were examined, taking about 10 minutes.
Distinction between bacilli and artifact: Among striking features of the bacilli examined by this technique is the clarity and reduced time duration with which their morphology is demonstrated in a clear background. This often makes their distinction from artifacts easy.
Age distribution of cases in the study(n=150)
Age in years
In the present study the age of the patients ranged from 12 months to 67 years. The maximum number of cases was in the age group of 21 to 30 years. There were 49 (32.6%) out of 150 cases in this age group.
Extremes of age were least affected. There were 12/150(8%) cases below 10 years and 14/150(9.3%) cases above 50 years.
II: Showing gender distribution (n=150)
Out of 150 cases , 71 were female and 79 were male. A male preponderance accounting for 79/150(52.6%) of cases was seen. The male to female ratio was 1:1.1
III: Showing the values of Erythrocyte sedimentation rate ESR (n=150)
ESR (mms/Ist hour)
Number of cases
0 to 15
16 to 30
31 to 45
46 to 60
Table is showing ESR values at the time of clinical presentation. The maximum number of cases (44%) had ESR range from >61mm/Ist hour. An appreciable increase in ESR (> 30 mm/Ist hour) was seen in 85.3% of cases.
IV: Comparison of conventional ZN method with modified bleach method for AFB detection
Direct ZN stain
Bleach ZN stain
The modified bleach method treatment of fine needle aspirates before ZN staining is able to detect more AFB as compared to conventional direct ZN stain. Bleach treated ZN smears had detected additional 62/150(41.66%) cases which were negative on direct ZN smears and lowered the AFB negative smear by 41.33%.
Tuberculosis is common in our country, and it is not surprising that TB lymphadenopathy continues to be one of the commonest causes of chronic lymph node enlargement, particularly in the cervical region. The diagnosis of tuberculosis is easy and simple when disease is florid or disseminated but localized involvement of extra- pulmonary organ or tissue may at times pose a diagnostic problem. The clinical parameters for the diagnosis of tuberculosis in lymph nodes are neither specific nor do their absence exclude tubercular involvement.
Early diagnosis of tuberculosis and initiating optimal treatment would not only enable a cure of an individual patient but will curb the transmission of infection and disease to others in community. In developing countries, microscopy of the specimen is by far the fastest, cheapest and most reliable method for the detection of AFB. Cytological diagnosis based on morphology and confirmation by a positive ZN stain for AFB is a time-tested method for the diagnosis of lymphadenitis.
The finding of necrosis cannot rule out the possibility of tuberculous lymphadenitis.Thus a purulent smear needs to be evaluating further. In present study all cases with purulent smear were subjected to Ziehl-Neelsen staining. Ergete et al126 (2000) suggested same thing in their study that Ziehl-Neelsen staining should be performed for confirmation and in particular for cases of abscess and necrosis as part of diagnostic step. Comparison of AFB positivity in direct ZN stained aspirate smears and bleach treated ZN smears with various studies:
AFB positivity in direct ZN stain
AFB positivity after BLEACH ZN
Gangane N et al114 (2008)
Vamseedhar Annam et al122 (2009)
Mandakini .M.Patel et al116(2013)
Chandrasekhar et al115(2012)
Gunja Dwivedi et al 117(2013)
Present study (2015)
The percentage of AFB positivity in the present study was marginally comparable to (63.3%) that reported in previous studies by Gangane et al.114 , Chandrasekhar et al115(2012) and Annam et al.122 (72% ,60.2%and 63.4%, respectively). The routine ZN stain has low sensitivity and in this study it was 22.3%, which is comparable to the range of other studies with lowest being 12.5% in study by Chandrasekhar et al115(2012) and highest in study by Gunja Dwivedi et al117(2013) 35.5% In the developing countries, the microscopy of the specimen is by far the fastest, cheapest and the most reliable methods for the detection of AFB. Cytological diagnosis based on morphology and confirmation by a positive ZN stain for AFB is a time-tested method for the diagnosis of tuberculous lymphadenitis.
Khubnani et al .131 studied 55 cases of extrapulmonary TB, which included 18 aspirates from body fluids, 18 from abscesses drained from various body sites, 17 from lymph nodes and two from skin scrapings. It was found that an overall 43.36% cases were suggestive of TB on cytology, 21.8% cases positive for AFB by conventional ZN staining and 70.90% cases positive for AFB by the bleach
method. In the present study of 150 cases, which included only lymphnode aspirates, TB was diagnosed in 37.33% on cytology, conventional ZN staining for AFB was positive in 22% and bleach method for AFB was positive in 63.66%.
In our study, all the samples which were positive for the direct smears were positive by the bleach method also. The results showed that there was a significant increase in the sensitivity with the use of 5% NaoCl. The increase in the 44.33% smear positivity with the use of 5% NaoCl with the centrifugation method was very encouraging as compared to that of the direct smears. Majority of the positive cases showed high AFB positivity that was making them easily visible and detectable, and the bacilli were seen in clumps in a thin background, making the screening process easier, faster and less strenuous on the eye. AFB morphology was observed to be better preserved in the bleach method, thus reducing the time required for examination of the slides for detection of AFB.
The present study validates the literature on the utility of the concentration of AFB by modified bleach method for detection of tuberculous bacilli in lymph node aspirates and is more sensitive than the conventional ZN method. Moreover, the modified bleach method is safe, easy to perform and inexpensive.
Limitations of the present study:
Patients receiving treatment were not excluded from study.
Haemorrhagic aspirates were excluded.
3. Other causes of the granulomatous inflammation such as fungus, foreign body, sarcoidosis were not excluded.
4. Confirmation by culture could not be done after bleach treatment.
In today’s world it is very well understood that clinician interest is in early and accurate diagnosis so that early treatment is started. The bleach method for AFB diagnosis is simple, safe and cost-effective and it does not require additional expertise.
2) The materials and reagents are affordable and they are available locally in the countries where TB is endemic.
3) The bleach used NaOCl is a potent disinfectant, also limiting the risk of laboratory-acquired infections.
4) The results would be more efficient if concentration by bleach solution,
relative centrifugal force and bleach treatment is as per the time schedule and is proportionate.
5) The implementation of the bleach method clearly improves microscopic
detection and can be a useful contribution to routine cytology.
6) AFB positivity grade was much higher with Bleach Method than with routine ZN staining, making bacilli easily visible, with shorter screening time.
7) This would be of benefit to the patients to receive an early and effective treatment.
Since NaOCl kills mycobacteria, this technique cannot be used for samples intended for culture, but the method can be used in laboratories that perform microscopy only.
This study suggests that the bleach method can significantly improve the diagnostic yield , 61.33% increased detection of AFB by microscopy for the TB diagnosis .Moreover, the modified bleach method is safe, easy to perform, faster and inexpensive method to diagnose a tuberculous lesion ,can be useful in routine cytology.
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