Tuberculous Lymphadenitis with Dark Specks Cytomorphology: Focus on the Presence of Eosinophils and History of Atopy in Children

Background. Research on eosinophils in lymph node cytology in tuberculous lymphadenitis (TBLN) patients with dark specks cytomorphology and its association with a history of atopies are still very little. Although neutrophils have been reported as predominant phagocytic cells in TB patients, eosinophils are also abundant in blood and lung tissue, but their role and pathogenesis are still unclear. Objective. To analyze the relationship between TBLN and eosinophils, dark specks, and history of atopy in pediatric TBLN patients. Methods. This study is a cross-sectional study of 35 indicative cytology results of patients with TBLN stained with May Grunwald Giemsa. Data on atopy history were measured by filling out the standard questionnaire of The International Study of Asthma and Allergies in Childhood (ISAAC). Results. Based on the cytological examination, 22 samples (62.9%) were found with positive eosinophils, and 13 samples (37.1%) with negative eosinophils. A total of 23 samples (65.7%) had a history of atopy. The Fisher's Exact Test results showed that TBLN with dark specks cytomorphology tends to find eosinophils with a test value of p=0.001, indicating a relationship between the two cytomorphological features. The results also showed a relationship between TBLN with dark specks cytomorphology with a history of allergies (p=0.038), and there was a relationship between the presence of eosinophils and a history of allergies (p=0.001). Conclusion. Tuberculous lymphadenitis with dark specks cytomorphology was significantly associated with eosinophils and a history of atopy.

Background. Research on eosinophils in lymph node cytology in tuberculous lymphadenitis (TBLN) patients with dark specks cytomorphology and its association with a history of atopies are still very little. Although neutrophils have been reported as predominant phagocytic cells in TB patients, eosinophils are also abundant in blood and lung tissue, but their role and pathogenesis are still unclear. Objective. To analyze the relationship between TBLN and eosinophils, dark specks, and history of atopy in pediatric TBLN patients. Methods. This study is a cross-sectional study of 35 indicative cytology results of patients with TBLN stained with May Grunwald Giemsa. Data on atopy history were measured by filling out the standard questionnaire of The International Study of Asthma and Allergies in Childhood (ISAAC). Results. Based on the cytological examination, 22 samples (62.9%) were found with positive eosinophils, and 13 samples (37.1%) with negative eosinophils. A total of 23 samples (65.7%) had a history of atopy. The Fisher's Exact Test results showed that TBLN with dark specks cytomorphology tends to find eosinophils with a test value of p=0.001, indicating a relationship between the two cytomorphological features. The results also showed a relationship between TBLN with dark specks cytomorphology with a history of allergies (p=0.038), and there was a relationship between the presence of eosinophils and a history of allergies (p=0.001). Conclusion. Tuberculous lymphadenitis with dark specks cytomorphology was significantly associated with eosinophils and a history of atopy. The incidence of extrapulmonary tuberculosis (EPTB) in children has been increasing rapidly in recent years. This disease can involve multiple organs, and the most common is the lymph nodes, known as tuberculous lymphadenitis (TBLN) (Bayazıt et al., 2004;Mustafa et al., 2005;Tadele et al., 2014;Gupta and Bhake, 2017;Shah and Dani, 2017).
Tuberculous lymphadenitis is caused by the reactivation of latent disease in a former lymph node infection or hematogenous reinfection. Signs and symptoms are chronic, painless swelling that persists and enlarges. The mass is known as a cold abscess because it is not accompanied by local discoloration and heat, and the overlying skin is purple (bluish-red) (Chao et al., 2002).
The diagnosis of TBLN is based on the typical cytologic predominantly reactive with few clusters of epithelioid cells, multinucleated giant cells, and necrotic material with few epithelioid cells. Lubis et al. (2008) found a different microscopic picture beyond the conventional picture of TBLN in the form of dark specks (DS) between eosinophilic granular amorphous necrotic masses. Dark specks are containing dark chocolate particles in pinkish, homogenous, acellular, and well-circumscribed irregular mass on H&E stain ( Figure 1) (Lubis et al., 2008). Prasoon (2014) and Chikkannaiah (2015) reported an eosinophilic structure similar to DS cytomorphology, associated with the presence of Acid Fast Bacillus (AFB) in TBLN with specificity values of 58.33% and 39% (Prasoon et al., 2014;Chikkannaiah et al., 2015). Delyuzar (2019) reported that positive TB cases (DS features) compared to negative TB cases (without DS features) were found to have a high specificity value (>90) (Delyuzar et al., 2019). Research conducted by Lubis et al. (2021) reported aspirating with dark specks that showed positive immunocytochemistry with rabbit polyclonal against Mycobacterium tuberculosis (Mtb) antibody (60%) . Therefore, the cytological appearance of DS is one of the diagnostic signs of TBLN and is used as an additional basis for establishing a cytological diagnosis of TBLN.
On TBLN with cytological appearance of DS, many eosinophils cytology was found, and requires pathological explanation. Eosinophils are also found in abundance in blood and lung tissue, but their role in its pathogenesis is still unclear. Eosinophilia in patients with pulmonary tuberculosis has been investigated in relation to the continued response to anti-TB chemotherapy and the initial allergic state of the organism (Kuzovkova et al., 2016).
Eosinophilia is associated with a cell-mediated T helper 2 (Th2) immune response, including the production of IL-5, which has the ability to degranulate and release cationic granule proteins that are helminthotoxic and function to increase eosinophilopoiesis and eosinophil activation (Weller and Spencer, 2018). So far, eosinophils have been associated with their function as host protection, but several studies have reported that the increase in eosinophils in helminth infections has not been shown to have a protective effect. Cytokines produced by eosinophils (including IL-10 and IL-4) suppress the host response and have no impact on larvae (Huang et al., 2014;Huang et al., 2015).
Similar to neutrophils, eosinophils can work to phagocytize pathogens that attack and kill them intracellularly by sending Major Basic Protein (MBP) and Eosinophil Cationic Protein (ECP) to intracellular phagosomes (Malik and Batra, 2012;Germic et al., 2019), thereby paving the way for subsequent antigen presentation (Shi et al., 2000). In addition, eosinophils also use extracellular killing mechanisms by releasing cytotoxins through degranulation, releasing droplets through Eosinophil Peroxidase (EPO), and trapping extracellular DNA (Svensson and Wennerås, 2005;Yousefi et al., 2008). Lubis (2019) reports increased expression of IL-4 cytokines on TBLN with DS feature .
Interleukin-4 plays an important role in the immune response to parasitic worms and allergic inflammation associated with atopy. Some researchers regard allergy as an imbalance to Th2, and recently immunologists have found a way to direct the Th2 allergic response toward amplification of the Th1 response to reduce the incidence of atopy. Several groups have tried to administer high doses of allergens to induce a Th1 response. Another group used the MTb vaccine to induce a stronger Th1 response at an early age (Berger, 2000).
In simple terms, to determine whether a person suffers from allergies or atopy is to see the presence of eosinophils in certain samples or by doing an Immunoglobulin E (IgE) examination. Eosinophils are a classic feature of infectious disease, and MTb infection frequently demonstrates the recruitment of eosinophils in naturally occurring human infections and experimental trials on animals. However, the precise function and role of eosinophils in the host immune response in TB are poorly understood. Several studies have stated that eosinophil protein is a mycobactericidal that can lyse MTb bacteria. In an in vitro study, EPO-induced surface changes were followed by MTb lysis, and EPO-containing macrophages exhibited strong antimycobacterial activity (Bohrer et al., 2021). It was also found that eosinophils can release defensins in response to cell wall components of BCG or MTb, which can directly kill BCG in vitro (Driss et al., 2009) This study was conducted to prove an increase in eosinophils in TBLN by the microscopic appearance of a DS necrotic mass and a history of atopy. This study is a preliminary study to establish whether there is a relationship between TBLN and the allergic process, especially in children, and an explanation of its pathogenesis so that treatment in TBLN is more focused on giving antiallergic drugs compared to anti-tuberculosis drugs, which cause many adverse side effects.

Sample Selection
This study was conducted using a cross-sectional approach to analyze the relationship between TBLN with eosinophils, dark specks, and atopy history in pediatric TBLN patients. The inclusion criteria of this study was the pediatric patients who have a lump that diagnosed as tuberculous lymphadenitis with DS who have a history of atopy. While the exclusion criteria were pediatric patients that diagnosed as non-specific infections and malignancies. Data on atopy history were measured by filling out the standard questionnaire of The International Study of Asthma and Allergies in Childhood (ISAAC). Cytology samples were obtained from private clinics and Muhammadiyah Hospital, North Sumatra. Fine needle aspiration biopsy was performed on the swollen neck, axillary, inguinal, supraclavicular, and submandibular lymph nodes in children aged 1-18 years. Microscopic examination was carried out at the Anatomical Pathology Laboratory, Faculty of Medicine, Universitas Muhammadiyah Sumatera Utara.

Cytological Assessment
Before the cytologic examination, a fine needle aspiration biopsy was performed with history taking and by filling standard questionnaire adapted from The International Study of Asthma and Allergies in Childhood (ISAAC). Aspirate slides were stained with May Grunwald Giemsa, and microscopic examination at 400x magnification was performed in 10 random areas. The diagnosis of tuberculous lymphadenitis was made based on a microscopic appearance of dark specks between eosinophilic granular amorphous necrotic masses (Lubis et al.,, 2008;Lubis et al., 2021), and the appearance of eosinophils in the form of a nucleus segmented into two or more lobes connected by thin filaments, abundant cytoplasm containing many reddishorange granules (Perkins et al., 2018). The slides were read by an anatomical pathologist using the blind method.

Statistical analysis
Descriptive statistics were used to present cytologic features of dark specks and eosinophils, and history of atopy. Fisher's exact test was employed to examine the relationship of TBLN with dark specks cytomorphology and eosinophils, the relationship between TBLN with dark specks cytomorphology with a history of allergies, and relationship between the presence of eosinophils and a history of allergies. All the analyzis conducted in this study used IBM SPSS 20.
The examination results for the cytomorphological diagnosis of dark specks among eosinophilic granular amorphous necrotic masses in TBLN, the presence of eosinophils, and a history of atopy are shown in Table 1. There were TBLN with dark specks cytomorphology in 23 cases (65.7%), positive eosinophils in 22 cases (62, 9%) (Figure 1), and a history of atopy in 23 cases (65.7%).  An analysis of the relationship between TBLN DS with a history of atopy and between eosinophils and a history of atopy was performed (Table 2). TBLN with DS tends to find eosinophils with a test value of p=0.001, indicating a relationship between the two cytomorphological features. The results also showed a relationship between TBLN and DS cytomorphology with a history of allergies (p=0.038), and there was a relationship between the presence of eosinophils and a history of allergies (p=0.001). This study showed an increase in eosinophils in field of view of the microscope. This study also showed a significant relationship between TBLN DS with a history of atopy and an increase in eosinophils with a history of atopy in pediatric patients. The specific immune response to MTb in the host is complex and involves several mechanisms. Chronic MTb infection creates an inflammatory environment that continuously develops in which to inhibit this development, the cellular response will be actively working. The cellular immune response in TB lasts 15-18 days after infection by involving dendritic cells that function to activate naive T cells, followed by the migration of activated T cells to the main site of infection. Inflammatory lesions are protected by the accumulation of activated T cells, macrophages, lymphocytes, and cytolytic-specific antigens. An increase in multifunctional T cells and a subset of functional T cells that produce the specific antigens interleukin (IL)-17, IL-22, and IL-23 were detected in MTb-infected individuals and is associated with an increase in granulocytes in TB granulomas, some of which are neutrophils and eosinophils (Caccamo et al., 2010;Clemmensen et al., 2021;Nogueira, Warren and Torres, 2021).
Eosinophils possess a large number of cell surface molecules, including Toll-like receptors, adhesion molecules, chemokines, complement, and chemotactic factor receptors, immunoglobulin receptors, apoptotic signaling molecules, prostaglandins, and leukotriene receptors (Barnig et al., 2015). These cells have complex extracellular and intracellular features that enable them to react to the inflammatory environment. Eosinophils store various preformed proteins, including cationic proteins, EPO and eosinophil-derived neurotoxins, cytokines, chemokines, and growth factors. Eosinophils exhibit degranulation via cytolytic degranulation in response to stimuli. Human eosinophils possess lipid bodies that can be induced at certain sites by forming eicosanoid mediators that are known to control bacterial infections (Vieira-De-Abreu et al., 2005).
The increase in eosinophils in almost the entire microscopic field of view is because eosinophils exhibit bactericidal potential mediated through phagocytosis, respiration, and mobilization of cytotoxic proteins in the presence of bacterial infection (Ramirez et al., 2018), which suggests a protective role of these cells in bacterial infection. Extensive necrotic in the form of amorphous eosinophilic granular mass is suspected to be a lipid body of eosinophils, which requires further examination. It can also be seen from the response to therapy given where in patients with a higher number of eosinophils with extensive necrotic masses, anti-tuberculosis treatment becomes more manageable and shows satisfactory results.
This study also showed a significant relationship between TBLN DS with a history of atopy and also an increase in eosinophils with a history of atopy in pediatric patients. It is obtained that children with a history of asthma are The history of atopy in this study was established based on the ISAAC questionnaire without conducting SPT and IgE level tests. Since the subject in this study was the local prevalence of TB, history of atopy, especially asthma, and socioeconomic status were relatively homogeneous. It was assumed that most subjects had initial exposure to infections and allergens. Further research will be conducted to prove whether there is a true relationship between TBLN and atopy history by using cytokine biomarkers to better understand the pathogenesis of this disease from an immunological perspective.
Research on the exact role and function of eosinophils in the host immune response to mycobacterial infection in TB remains should be carried out comprehensively both experimentally in experimental animals and clinically. The involvement of eosinophils and a history of atopy in the study were initial findings, suggesting the possibility of coinfection between TB and allergy. Antiallergic drugs can be considered in future management strategies apart from relying on BCG and ATT, but further research is needed. The extensive analysis and correlation between eosinophilia and TB represent a further goal to better understand the role of eosinophils during TB infection and disease progression.

LIMITATION OF THE STUDY
Since this study was conducted using a cross-sectional approach to analyze the relationship between TBLN with eosinophils, dark specks, and atopy history in pediatric, there was limited variables and indicators, especially related to confirmation by standard examination of atopy determination with IgE. Future studies will be more full of insights if consider broader indicators to definitive diagnosis of atopy.

CONCLUSION AND SUGGESTIONS
Tuberculous lymphadenitis with dark specks cytomorphology was significantly associated with eosinophils, and a history of atopy. This study can be considered to that treatment in TBLN is more focused on giving anti-allergic drugs compared to anti-tuberculosis drugs, which cause many adverse side effects.