| Abstract|| |
Background: Acid-fast bacilli (AFB) is not seen in all necrotic tuberculous lesions. If the subset of tuberculous lesions which yield positive result for AFB can be identified, it would save on time and manpower besides optimizing use of resources. A prospective study was undertaken to assess if presence of eosinophilic structures (ESs) in necrotic tuberculous lesions correlated with the presence of AFB.
Materials and Methods: Patients referred for fine needle aspiration cytology for evaluation of lymphadenopathy between July 2012 and June 2013 were analyzed. The hematoxylin and eosin and May-Grünwald-Giemsa stained slides were screened for epithelioid cell granuloma, ES and necrosis and Ziehl Neelsen stained smears for AFB.
Result: One hundred and eight tuberculous lymph nodes yielded necrotic material on aspiration. Four cytologic pictures were seen: (a) ES + AFB + in 58.33%, (b) ES + AFB− in 20.37%, (c) ES− AFB + in 9.26% (d) ES− AFB− in 12.04% cases. Overall AFB was found in 67.59% cases, out of which 58.33% correlated with the presence of ES while 9.26% were seen in smears without ES.
Conclusion: Presence of ESs should be included in the morphological description of tuberculous lesions. In the absence of granulomas, they indicate tuberculous nature of the lesion. Presence of ES mandates a search for AFB as probability of finding AFB is high in such lesions. Significance of ES lies in their presence and not in their absence. Eosinophilic structures appear to be the missing link in the spectrum of tuberculous lesion.
Keywords: Acid fast bacilli; eosinophilic structures; fine needle aspiration cytology; tuberculosis
|How to cite this article:|
Prasoon D, Agrawal P. Correlation of eosinophilic structures with detection of acid-fast bacilli in fine needle aspiration smears from tuberculous lymph nodes: Is eosinophilic structure the missing link in spectrum of tuberculous lesion?. J Cytol 2014;31:149-53
|How to cite this URL:|
Prasoon D, Agrawal P. Correlation of eosinophilic structures with detection of acid-fast bacilli in fine needle aspiration smears from tuberculous lymph nodes: Is eosinophilic structure the missing link in spectrum of tuberculous lesion?. J Cytol [serial online] 2014 [cited 2020 Feb 27];31:149-53. Available from: http://www.jcytol.org/text.asp?2014/31/3/149/145647
| Introduction|| |
Fine needle aspiration cytology (FNAC) is commonly used for diagnosis of tuberculosis. Cytodiagnosis of tuberculosis involves the identification of epithelioid cell granuloma, necrosis and acid-fast bacilli (AFB) in the smears. While identification of epithelioid cell granuloma and necrosis help in the diagnosis of tuberculosis, identification of AFB provides etiological evidence of tuberculosis. Thus, all effort should be made to identify AFB in FNA smears.
Identification of AFB can be done using Ziehl Neelsen (ZN) stain,  fluorescent stain,  mycobacterial culture,  or by nucleic acid amplification.  Identification using fluorescent stain, culture or nucleic acid amplification techniques are effective, but require specialized infrastructure and training besides being expensive. Thus, ZN stain is still widely used for identification of AFB in FNA smears.
It is commonly known that AFB is not seen in all tuberculous lesions. It is also known that AFB is more often encountered in necrotic lesions.  It is also known that all necrotic tuberculous lesions do not yield AFB on microscopy. 
Granuloma and necrosis can be identified either under low-power or high-power objectives. However, identification of AFB requires searching under the oil immersion lens and so needs time, effort and motivation. Thus, if some morphological change in tuberculous smears could be identified, whose presence correlated with the presence of AFB, it would save on time and manpower besides optimizing the use of resources. With this in view, a prospective study was undertaken to assess if presence of eosinophilic structures (ES)  in smears from necrotic aspirates in tuberculous lesions correlated with the presence of AFB in that lesion.
| Materials and Methods|| |
Patients referred for FNAC for evaluation of their lymphadenopathy between July 2012, and June 2013 were analyzed. Only those lymph nodes which yielded necrotic material on aspiration, and were clinically and therapeutically proven to have tuberculosis were included in this study. The sample size was determined by the period of study and the inclusion criteria detailed above. Most patients did not show evidence of tuberculosis at any other site. No case of atypical mycobacteriosis was encountered. Furthermore, none of the patients were HIV positive nor was any patient immunocompromised or receiving immunosuppressive therapy. Each patient was thoroughly examined. Data regarding age, sex and lymph node involved were noted.
Non suction method was mostly used for collecting the sample. However, whenever required aspiration was done using 22 gauge needle and 10 mL syringe. Care was taken not to produce a sinus by aspirating from the nondependent area. Gross appearance of the aspirate was noted in each case.
Three smears were made in each case. One slide was stained with hematoxylin and eosin (H and E) stain, second with May-Grünwald-Giemsa (MGG) stain and third with ZN stain. If ZN stain was unsatisfactory or if need for more screening was felt, the slide with MGG stain was decolorized with 1% acid alcohol and restained with ZN stain. All necrotic aspirates were subjected to mycobacterial culture and biochemical tests for exclusion of atypical mycobacteriosis.
Eosinophilic structures appear as irregular, homogenous, acellular structures with well defined margins and appear eosinophilic in H and E stain, basophilic in MGG stain and bluish in ZN stain.
We have not observed similar ESs in any other disease. Hence, a negative control could not be set up in this study.
Statistical analysis to evaluate the association between the two variables, viz, ES and incidence of AFB detection was done using Chi-square test with Yates correction.
| Results|| |
During the study period, 108 tuberculous lymph nodes yielded necrotic material on aspiration. The age group most commonly involved was 11-30 years. It was uncommon over 40 years of age. Females were more commonly involved in a ratio of 1.3:1. Cervical, axillary and inguinal lymph nodes were involved in various combinations. The most common presentation was as unilateral cervical lymphadenopathy.
Eosinophilic structures were identified in H and E, MGG and ZN stained smears. However, they were best appreciated in the H and E stained smears [Figure 1]. In relation to ESs and AFB, four different cytologic pictures were seen [Table 1].
|Figure 1: Eosinophilic structure surrounded by necrotic material (H and E, ×200)|
Click here to view
- Eosinophilic structures + , AFB +
- Eosinophilic structures + , AFB−
- Eosinophilic structures− , AFB +
- Eosinophilic structures− , AFB-
Eosinophilic structure + , acid fast bacilli +
This was the most common picture and was seen in 63 cases (58.33%). AFB was seen within the ESs in 11 cases and outside ESs in 47 cases. AFB was seen both within and outside ESs in 5 cases. Interestingly, epithelioid cell granulomas were seen in only 28 out of these 63 cases.
Eosinophilic structure + , acid fast bacilli−
This picture was seen in 22 cases (20.37%). All the 22 cases showed epithelioid cell granulomas.
Eosinophilic structure− , acid fast bacilli +
This picture was seen in 10 cases (9.26%). 3 out of these 10 cases showed epithelioid cell granulomas.
Eosinophilic structure− , acid fast bacilli−
This picture was seen in 13 cases (12.04%). Epithelioid cell granulomas were seen in all the 13 cases, and their presence suggested the diagnosis of tuberculosis. A few more cases were seen with this picture where no granulomas were seen. These could not be labeled cytologically as tuberculous and hence were not included in this study. However, follow-up showed that several of these patients responded to anti-tuberculous treatment.
Of 108 necrotic tuberculous aspirates, 85 cases showed ESs. Out of these 85 cases, AFB was seen in 63 cases (63/108 = 58.33%). On the other hand, there were 23 smears which did not show ESs. Out of these 23 smears AFB was found in 10 cases (10/108 = 9.26%). Overall AFB was seen in 73 out of 108 cases (67.59%). Furthermore, AFB was more often seen outside the ESs than within them.
Chi-square test with Yates correction was applied to evaluate the association between the two variables - ES and incidence of AFB detection. χ2 = 6.422 with 1 degrees of freedom. The P = 0.0113. Thus, a statistically significant association was observed between ES and incidence of AFB detection.
| Discussion|| |
Eosinophilic structures, as described by Pandit et al.,  are degenerated acellular granulomas. They appear in H and E stained smears as pink, irregular, homogenous, acellular structures with well-defined margins. They are distinct from the surrounding eosinophilic necrotic material from which they can be readily differentiated [Figure 1]. They have the following features:
a. Morphologically resemble degenerating granulomas, but are acellular.
b. Reveal the presence of mycobacterial antigen by immunohistochemical staining
c. Are always associated with caseous necrosis
d. Are not seen in non tuberculous necrotic lesions. 
Delayed type hypersensitivity reaction is the principal pattern of immunologic response to intracellular microbiologic agents like Mycobacterium tuberculosis.  About 3 weeks after infection, a Th1 response against M. tuberculosis is mounted. Secretion of Th1 cluster of cytokines, particularly gamma interferon, activate macrophages, the ability of which to phagocytose and kill the micro-organism is markedly augmented. These activated macrophages aggregate around the lesion's center and effectively neutralize tubercle bacilli without causing further tissue destruction. In the central part of the lesion, the necrotic material assumes the aspect of caseous necrosis. 
When the macrophage activating response is weak, mycobacterial growth can be inhibited only by intensified delayed type hypersensitivity reaction, which lead to tissue destruction. The lesion tends to enlarge further, and the surrounding tissue is progressively damaged. At the center of the lesion caseous material liquefies. This liquefied caseous material contains a large number of bacilli. 
It would not be unreasonable to argue that as necrosis continues, not only does caseous material liquefy, but granulomas also degenerate to assume the appearance of acellular ESs. Thus, it may be deduced that ESs would be seen in that phase of the disease when the necrotic lesions are rich in AFB.
The cytologic picture seen in tuberculosis depends upon the host's immune response and the stage of disease at the time of aspiration. The four cytologic pictures detailed above are different stages in the evolution of the disease. The picture of ES - AFB - relates to that stage of disease where the granulomatous response is adequate in containing the infection and necrosis is minimal. As the disease progresses, the granulomas begin to degenerate to form ESs, though they are still able to contain AFB, giving rise to the picture ES + AFB− . The picture of ES + AFB + relates to that phase where disease has progressed a step further, and the granulomas are unable to contain the infection and have degenerated to form ESs. Tissue destruction has progressed, and the lesion is rich in mycobacteria. It is in this stage that we have encountered the highest AFB positivity in this study. The picture of ES - AFB + relates to a stage still further in evolution of the disease. Here the bacteria overwhelm the granulomatous response and necrosis is more developed. As the disease progresses necrosis becomes more widespread, but growth of mycobacteria gets inhibited by low-oxygen tension and low-pH created by the necrotic environment itself. The necrotic smears which did not show either granuloma or ES or AFB relate to this phase.
Using ES as an indicator for the presence of AFB in necrotic aspirates from tuberculous lymph nodes, AFB was detected in 63 cases (63/108 = 58.33%). However, 22 cases showing ESs did not show AFB. All these 22 cases showed epithelioid cell granulomas, fortifying the view that the relation between the presence of granuloma and of AFB is inverse.  Moreover, AFB was seen in 10 cases that did not show ES. Thus, the significance of ES lies in their presence and not in their absence.
In tuberculous FNA smears AFB positivity has been reported from 22.7% to 56.92% on ZN stain, ,,,,,,, 45.4-98.33% on fluorescent stain, , 46.0-82.8% on culture, ,, and 55.9-94.6% on PCR. ,, In our study AFB, positivity on ZN stain was found to be 67.59% out of which 58.33% correlated with the presence of ES.
Like in leprosy, there seems to be an immunological gradient in tuberculosis also. At one end of the spectrum, when the host immune response is good, there are well-formed granulomas without ESs or necrosis or AFB. At the other end of the spectrum, when the host immune response is poor, lesions are purely necrotic without granulomas or eosinphilic structures or AFB. In between lie stages with degenerating granulomas, ESs and purely necrotic lesions with AFB. The presence of AFB in smears depends upon several factors like presence of granulomas, necrosis and pH and pO 2 of the lesions. The cutoff point for finding AFB in the above mentioned gradient is not yet known. There may be a quantitative threshold value for granulomas below which AFB are found in the smears. At the other end of the spectrum, the pH and pO 2 of the lesion may be the factors that determine if AFB would remain viable in the necrotic environment. It is known that tubercle bacilli are strict aerobes and thrive at a pO 2 of 140 mm Hg and become scarcer in necrotic tissue lacking blood-borne oxygen.  Their growth is also inhibited by a pH lower than 6.5.  Analysis of these parameters may provide us with a frame within which AFB would be detected in cytology smears.
| Conclusion|| |
As of now, we feel the following conclusions can be drawn from the above study:
- Presence of ESs should be included in the morphological description of tuberculous lesions. In the absence of granulomas, they indicate tuberculous nature of the lesion.
- Presence of ES mandates a search for AFB as the probability of finding AFB is high in such lesions.
- Significance of ES lies in their presence and not in their absence.
- Eosinophilic structures appear to be the missing link in the spectrum of tuberculous lesion which appears to be a continuum extending from one end with purely granulomatous lesion, through degenerating granuloma, acellular ES, necrotic lesions with AFB to finally necrotic lesion without AFB at the other end.
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