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IDENTIFICATION OF ACID FAST BACILLI IN GRANULOMATOUS LYMPH NODE TISSUE BIOPSY.

Byline: Ghulam Rasool, Usman Ali, Farman Ali, Nadia Naseem and A.H Nagi

ABSTRACT

Background: Tuberculous lymphadenitis is a common form of extrapulmonary tuberculosis in young adults and children which commonly involves superficial lymph nodes. The purpose of this study was to detect and confirm the presence of acid fast bacilli in lymph node biopsies using routine Ziehl Neelsen stain, Kinyoun stain and fluorescence staining and also to determine the best, rapid and more cost effective technique of staining for the detection of acid fast bacilli.

Material and Methods: This descriptive cross-sectional study was conducted in University of Health Sciences, Lahore, Punjab, Pakistan, from November, 2013 to February 2014. Sample size was 97 patients with lymph node biopsies taken from through non probability, consecutive sampling technique. Demographic variables were; gender, age group. Research variables were; site of lymph node biopsy, type of granulomatous inflammation, positivity of AFB. The attributes of age group were; 1-15. 16-30, 31-45 and 46-75. The attributes of site of lymph node biopsy were; supra-clavicular, cervical, axillary, right mediastinal and submandibular. The attributes of type of granulomatous inflammation were; caseous and non-caseous and of positivity of AFB were; positive and negative. Data was collected in Gulab Devi Chest Hospital, Lahore. Lymph node biopsies from different sites. including supraclavicular, cervical, axillary, right mediastinal and Submandibular were included and stained with. Descriptive analysis was done.

Results: Out of a total of 97 patients with granulomatous lymph node biopsies, 36 (37%) were males and 61 (63%) females. Caseous granulomas were 67(69%), while 30(31%) showed non-caseous granulomas. Lymph nodes were collected from different sites e.g. supraclavicular (20, 21%), cervical (69, 71%), axillary (4, 4%), right mediastinal (2, 2%) and Submandibular (2, 2%) lymph nodes. AFB were microscopically positive in 54 (56%) nodal tissue biopsy sections and forty three (44%) cases were negative. From a total of 54 positive cases routine ZN stain was positive in 45 (46%), Kinyoun stain was positive in 54 (56%) and fluorescent staining was positive in 54 (56%) cases.

Conclusion: Caseous granulomas were detected in females especially. Kinyoun and Fluorescent staining using Auramine O and Rhodamine B are better staining techniques in the detection of AFB than Ziehl Neelsen Staining.

KEY WORDS: Haematoxylin and eosin; acid fast bacilli; Kinyoun; Ziehl Neelsen; Lymph node.

INTRODUCTION:

Lymph node involvement by tuberculosis is the most common form of extrapulmonary tuberculosis. Tuberculous lymphadenitis constitutes 20 - 40% of extrapulmonary tuberculosis.1 Tuberculosis commonly involves superficial lymph nodes including those in posterior and anterior cervical chains or the suprascapular fossae and others like submandibular, periauricular, inguinal and axillary groups may also be involved.2

Mycobacterium tuberculosis detection staining technique by fluorescence microscopy is about 10% more sensitive than the Ziehl-Neelsen staining technique and has a similar specificity. However fluorescent staining is technically more complicated.3

Kinyoun observed that fluorescence microscopy was expensive than routine Ziehl-Neelsen staining and stain is also performed in a few minutes.4 Fluorescence microscopy increases the number of samples that can be read in a given time, because more slides can be stained and seen under the microscope and therefore is expensive than Ziehl-Neelsen stain.5

The objective of this study was to detect and confirm the presence of acid fast bacilli in lymph node biopsies using routine Ziehl Neelsen stain, Kinyoun stain and fluorescence staining and also to determine the best, rapid and more cost effective technique of staining for the detection of acid fast bacilli.

MATERIAL AND METHODS:

This descriptive cross-sectional study was conducted in University of Health Sciences, Lahore, Punjab, Pakistan, from November, 2013 to February 2014. Sample size was 97 patients with lymph node biopsies taken from through non probability, consecutive sampling technique. Demographic variables were; gender, age group. Research variables were; site of lymph node biopsy, type of granulomatous inflammation, positivity of AFB. The attributes of age group were; 1-15. 16-30, 31-45 and 46-75. The attributes of site of lymph node biopsy were; supra-clavicular, cervical, axillary, right mediastinal and submandibular. The attributes of type of granulomatous inflammation were; caseous and non-caseous and of positivity of AFB were; positive and negative. Data was collected in Gulab Devi Chest Hospital, Lahore.

Lymph node biopsies from different sites including supraclavicular, cervical, axillary, right mediastinal and Submandibular were included and stained with H and E to determine the caseous or noncaseous granulomas. Slides were stained with routine ZN, Kinyoun and fluorescent staining technique using Auramine O and Rhodamine B to demonstrate the presence of acid fast bacilli in tissue. The tissue was removed from patient and fixed in 10 % formalin. Then the tissue was processed by automatic tissue processor Microm GmbH cat. No."813150" using different reagents (10 % formalin, ethanol, xylene and paraffin wax). After this tissue was embedded in automatic Tissue Tek TEC model "TEC 5 EMJ-2" using molten paraffin wax. Paraffin embedded sections were prepared using microtome model "RM 2125 RT". The H and E stain was performed on the slides to determine the histological diagnosis for selecting tissues and only the caseous or noncaseous granulomatous lymph nodes were selected.

Slides were stained with routine ZN staining, Kinyoun staining and fluorescent staining technique using Auramine O and Rhodamine B to demonstrate the acid fast bacilli in tissue.

For the detection of AFB from three stains, the study was carried out in four phases. In the first phase, all 97 lymph node biopsies were stained and positive and negative cases were separated. In phase 2, the negative cases were recut at deeper levels and stained with all 3 stains. Again positive and negative cases were separated and the negative cases of this phase were recut at further deeper levels and stained with all three stains in phase 3 till we got all negative cases by further re-cutting and staining in phase 4. Positive and negative controls were run for quality control of H and E staining, ZN staining, Kinyoun staining and fluorescent staining. Results of routine ZN and Kinyoun staining demonstrated as AFB red and background was blue. Results of fluorescence staining technique demonstrated AFB as green (using fluorescence 530 nm) while the background was black. All the variables were categorical. Frequencies and percentages were calculated. Descriptive analysis was done.

RESULTS:

Out of a total of 97 patients with granulomatous lymph node biopsies, 36 (37%) were males and 61 (63%) females. The male to female ratio was 1: 1.7. Out of a total of 97 patients with granulomatous lymph node biopsies, caseous granulomas were 67 (69%), while 30 (31%) showed non-caseous granulomas. Lymph nodes were collected from different sites e.g. supraclavicular (20, 21%), cervical (69, 71%), axillary (4, 4%), right mediastinal (2, 2%) and submandibular (2, 2%) lymph nodes (Table 1).

Table: 1 Multi variate descriptive analysis of gender, age group, type of Granulomatous inflammation and Site of lymph node biopsy (n=97)

###attributes###Caseous Granuloma###Non-caseous Granuloma###Total (97)

###67(69%)###30(31%)

###Male###21(22%)###15(15%)###36(37%)

Gender

###Female###46(48%)###15(%)###61(63%)

###01 - 15###15(16%)###6(6%)###21(22%)

###16 - 30###35(36%)###15(15%)###50(51%)

Age group

###31 - 45###14(14%)###8(8%)###22(23%)

###46 - 75###3(3%)###1(1%)###04(04%)

###Supraclavicular###14(14%)###6(6%)###20(21%)

###Cervical###47(48%)###22(23%)###69(71%)

Site of lymph

###Axillary###2(2%)###2(2%)###4(4%)

node biopsy

###Right mediastinal###2(2%)###0(0%)###2(2%)

###Submandibular###2(2%)###0(0%)###2(2%)

Table: 2 Frequencies of positivity of AFB as stained by ZN, Kinyoun and Flourescent staining in 4 phases of the study (n=97)

###Positive AFB (%)###Total Positive###Total Negative

Phase###Total L.N biopsies###L.N Biopsies###L.N Biopsies

###ZN###Kinyoun###Flourescent###(%)###(%)

I###97###35(36)###40(41)###40(41)###40(41)###57(59)

II###57###7(12)###9(16)###9(16)###49(51)###48(49)

III###48###3(6)###5(10)###5(10)

IV###43###0###0###0

###54(56)###43(44)

Total###45(46)###54(56)###54(56)

Out of a total of 97 granulomatous lymph nodes, AFB were microscopically positive in 54 (56%) nodal tissue biopsy sections and forty three (44%) cases were negative. From a total of 54 positive cases routine ZN stain was positive in 45 (46%), Kinyoun stain was positive in 54 (56%) and fluorescent staining was positive in 54 (56%) cases. The three stains were positive in 45 (46%) cases. (Table 2).

In phase I, all the three stains i.e. ZN, Kinyoun and fluorescent stains were positive for AFB in 35 (36%) biopsies. Kinyoun staining was positive in 40 (41%) and fluorescent stain was positive in 40 (41%) cases.

In phase II, rest 57 negative biopsies were recut at deeper levels and stained with all three stains to demonstrate acid fast bacilli. Nine (16%) cases were positive for acid fast bacilli while 48 (49%) were negative for any staining.

In phase III, the remaining 48 (49%) cases which were negative for AFB were taken and their paraffin embedded blocks were recut 3rd time. Their sections were stained with all three stains to demonstrate acid fast bacilli. Further 05 (10%) positive cases for acid fast bacilli were seen.

In phase IV, the remaining 43 (44%) cases which were negative for AFB in phase 3 were taken and their paraffin embedded blocks were recut 4th time and stained with all three stains to demonstrate acid fast bacilli and no positive case for acid fast bacilli was seen this time.

DISCUSSION:

Routine ZN stain was frequently used by various workers.6,7 Study conducted by Krishnaswammi and Job in 1972 reported 91 of 128 (71%) AFB positive lymph nodes having tuberculous lymphadenitis after ZN stain.8 Greenwood and Fox in 1973 on "a comparison of methods for staining tubercle bacilli in histological sections" showed 33 out of 70 (47%) tuberculous morphology cases being positive for AFB on ZN staining.9 Eshete and colleagues in 2011 reported 37 of 60 (62%) lymph nodes with tuberculous histology being positive for AFB after ZN staining.10 In the present study, 45 of 97 lymph nodes biopsies were positive for AFB for all the three stains. From a total of 54 positive cases, routine ZN was positive in 45 (46%) cases from whom 21 (22%) were males and 30 (31%) were females. Presence of AFB in 45 (46%) cases among 97 showed that ZN staining was confirmatory as compared to H and E staining for AFB in lymph node tuberculosis.10 Our study revealed 46 % positive results for routine ZN staining technique.

Kinyoun staining technique is also known as cold Ziehl-Neelsen staining technique (1915). This is considered as 10 % better than Ziehl-Neelsen staining technique.4 In this study, of 97 lymph node biopsies, 45 were positive for all the three stains and Kinyoun stain was positive in 54 (56%) cases.

Fluorescent staining technique is better than routine Ziehl-Neelsen staining and cold Ziehl-Neelsen (Kinyoun) staining techniques. Study conducted by Krishnaswammi and Job in 1972 on "The role of ZN and fluorescent stains in tissue sections in the diagnosis of tuberculosis" showed 79.7 % positivity for AFB on fluorescent technique. They used the Kuper and May (1960) method by using Auramine O and Rhodamine B.8 Study conducted by Greenwood and Fox in 1973 on "A comparison of methods for staining tubercle bacilli in histological sections" showed 42 (60%) positive AFB on fluorescent technique out of 70 tuberculosis cases. They applied the Mansfield (1970) method by using Auramine O and phenol.9 In the present study, 45 lymph node biopsies were positive for three stains and fluorescent stain was positive in 54 (56%) cases from which 22 (23%) were males and 32 (33%) were females. Our study revealed 56 % results for fluorescent staining technique.

Various workers used the Ziehl-Neelsen and fluorescent stain for the investigation of smear 11-14

However there are only a few who used the tissue sections.11,12 Some workers have shown in their reports that fluorescent staining technique is superior to Ziehl-Neelsen staining technique 11,13 whereas others claim that fluorescent staining technique had equivalent results with Ziehl-Neelsen staining technique.14

Our study is comparable with the study of Krishnaswammi and Job in 1972 on "The role of ZN and fluorescent stains in tissue sections in the diagnosis of tuberculosis", Greenwood and Fox in 1973 on "A comparison of methods for staining tubercle bacilli in histological sections" and Eshete and others in 2011 "M. tuberculosis in lymph node biopsy paraffin embedded sections". However, in the present study, Kinyoun stain is used also and its results are equivalent to fluorescent staining technique.

CONCLUSIONS

Caseous granulomas were detected in females especially. Kinyoun and Fluorescent staining using Auramine O and Rhodamine B are better staining techniques in the detection of AFB than Ziehl Neelsen Staining. It is recommended that if the section is negative for acid fast bacilli using any stain, further deeper cuts of sections helps us to find the acid fast bacilli.

REFERENCES

1. Fangrat A, Domagala-Kulawik J, Krenke R, Safianowska A, Walkiewicz R, Chazan R. Diagnosis of tuberculous lymphadenitis based on the fine needle aspiration samples analysis. Pneumonologia i alergologia polska 2005;74: 126-8.

2. Gupta. Difficulties in the management of lymph node tuberculosis 2004; Lung India 2004;21: 50-3

3. Steingart KR, Henry M, Ng V, Hopewell PC, Ramsay A, Cunningham J, et al. Fluorescence versus conventional sputum smear microscopy for tuberculosis: a systematic review. The Lancet Infect Dis 2006;6: 570-81.

4. Kivihya-Ndugga L, van Cleeff M, Juma E, Kimwomi J, Githui W, Oskam L, et al. Comparison of PCR with the routine procedure for diagnosis of tuberculosis in a population with high prevalences of tuberculosis and human immunodeficiency virus. J Clin Microb 2004;42: 1012-5.

5. Reid MJ, Shah NS. Approaches to tuberculosis screening and diagnosis in people with HIV in resource-limited settings. The Lancet Infect Dis 2009;9: 173-84.

6. Mackellar A, Hilton HB, Masters PL. Mycobacterial lymphadenitis in childhood Arch dis in childhood 1967;42: 70.

7. Reid JD, Wolinsky E. Histopathology of Lymphadenitis Caused by Atypical Mycobacteria 1, 2. Am Rev Respir Dis 1969;99: 8-12.

8. Krishnaswami H, Koshi G, Kulkarni KG, Job CK. Tuberculous lymphadenitis in South India-a histopathological and bacteriological study. Tubercle. 1972;53: 215-20.

9. Greenwood N, Fox H. A comparison of methods for staining tubercle bacilli in histological sections. J Clin Pathol 1973;26: 253-7.

10. Eshete A, Zeyinudin A, Ali S, Abera S, Mohammed M. M. tuberculosis in lymph node biopsy Paraffin-embedded sections. Tuberle Res and treatment 2011;2011.

11. Koch ML, Cote RA. Comparison of fluorescence microscopy with Ziehl-Neelsen stain for demonstration of acid-fast bacilli in smear preparations and tissue sections. Am Rev Respir Dis 1965;91: 283-4.

12. Braunstein H, Adriano SM. Fluorescent Stain for Tubercle Bacilli in Histologie Sections I. Diagnostic Efficiency in Granulomatous Lesions of Lymph Nodes. Jr Am Clin Pathol 1961;36: 37-40.

13. Yamaguchi Jr BT, Braunstein H. Fluorescent Stain for Tubercle Bacilli in Histologic Sections. II. Diagnostic Efficiency in Granulomatous Lesions of the Liver. Jr Am Clin Pathol 1965;43: 184-7.

14. Weiser OL, Sproat EF, Hakes JD, Morse WC. Fluorochrome staining of mycobacteria. Jr Am Clin Pathol 1966;46: 587-8.
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Publication:Gomal Journal of Medical Sciences
Article Type:Report
Geographic Code:9PAKI
Date:Dec 31, 2016
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