Tuberculosis Revisited: Cytological Perspective Debajyoti Chatterjee, M.B.B.S., M.D.1 and Pranab Dey, M.D., M.I.A.C., F.R.C.Path2*
Tuberculosis is a major public health problem, especially in developing countries. The clinical manifestations of tuberculosis are very nonspecific and may mimic many other conditions. An accurate diagnosis is extremely important as the disease is treatable with antituberculous therapy. Cytological examination can provide useful diagnostic material for routine and ancillary techniques for rapid and accurate diagnosis of tuberculosis. Here we review the different morphological patterns of tuberculosis, challenges, and application of ancillary techniques for cytological diagnosis of tuberculosis. Diagn. Cytopathol. 2014;42:993–1001. VC 2014 Wiley Periodicals, Inc. Key Words:
tuberculosis; cytology; FNAC; lymph node; PCR
Introduction Tuberculosis is a major public health problem, especially in developing countries. According to the WHO Global tuberculosis report of 2012, the overall estimated global incidence of tuberculosis is 8.7 million, half of which is shared by India and China.1 In the western world, there is again a resurgence of tuberculosis because of HIV coinfection. Tuberculosis remains the major cause of death among AIDS patients, with up to 29% showing central nervous system (CNS) involvement.2 Pulmonary tuberculosis is the commonest form of disease. Among the extra-pulmonary locations, tuberculous lymphadenitis is the most frequent. Central nervous system involvement is seen around 10% of tuberculosis patients and represents the most serious legacy.3 Tuberculosis is caused by Mycobacterium tuberculosis, which is an aerobic, nonmotile and noncapsulated bacterium. Mycobacteria resist decolourisation by dilute
1 Department of Pathology, Post Graduate Institute of Medical Education and Research, PGIMER, Chandigarh, India 2 Department of Cytology, Post Graduate Institute of Medical Education and Research, Chandigarh, India *Correspondence to: Dr Pranab Dey, MD, MIAC, FRCPath, Department of Cytology, Postgraduate Institute of Medical Education and Research, Chandigarh, India. E-mail: [email protected] Received 30 December 2013; Accepted 11 June 2014 DOI: 10.1002/dc.23190 Published online 27 June 2014 in Wiley Online Library (wileyonlinelibrary.com).
C 2014 WILEY PERIODICALS, INC. V
mineral acid; therefore, mycobacteria are called “acid fast bacilli” or AFB. The term M. tuberculosis complex includes M. tuberculosis, M. bovis, M. africanum, and M. microti. M. microti does not cause human disease.4 Atypical mycobacteria, also known as “nontuberculous mycobacteria” (NTM) cause opportunistic infections in immunocompromized hosts. They include M. avium, M. intracellulare, M. kansasii, M. marinum, etc. Morphologically they cannot be distinguished from M. tuberculosis. This distinction is important as treatment of NTM infection is different from tuberculosis.5 The clinical manifestations of tuberculosis are very nonspecific and may mimic many other conditions. An accurate diagnosis is extremely important as the disease is treatable with antitubercular therapy. Cytological examination can provide useful diagnostic material for routine practice and ancillary techniques for rapid and accurate diagnosis of tuberculosis. Here we review the different morphological pattern of tuberculosis, challenges, and application of ancillary techniques for cytological diagnosis of tuberculosis.
Clinical Manifestations: Pulmonary Tuberculosis Lung is the primary site of infection by M. tuberculosis. Pulmonary tuberculosis may be primary or post-primary (secondary). Primary tuberculosis and progressive primary tuberculosis is seen mainly in children. Post primary tuberculosis is usually a disease of adult life. Primary tuberculosis is characterized by granulomatous inflammation. The main pathology of post primary tuberculosis is lipoid pneumonia, followed by caseous pneumonia and fibrocavitary lesion.6 Pulmonary tuberculosis is usually diagnosed by sputum microscopy for Acid fast bacilli (AFB), accompanied by culture or molecular techniques. The role of fine-needle aspiration cytology (FNAC) is limited in the diagnosis of pulmonary tuberculosis. Sputum examination for AFB is an integral part of global TB control strategy, and usually done on 3 consecutive days. Usually sputum screening for AFB is not done by cytologists. Sputum microscopy is a very cost effective and simple test but lacks sensitivity, especially when the bacterial concentration in sputum Diagnostic Cytopathology, Vol. 42, No 11
993
Diagnostic Cytopathology DOI 10.1002/dc
CHATTERJEE AND DEY
Fig. 1. Epithelioid cell granulomas along with large number of inflammatory cells. (May Grunwald Giemsa 3 higher magnification). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary. com.]
Fig. 2. Necrosis in tuberculosis (May Grunwald Giemsa 3 higher magnification). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
is less than 10,000/mL. The sensitivity can be increased by use of fluorescent microscopy.7 For children where sputum is difficult to obtain, gastric lavage for AFB is used as a screening modality for tuberculosis. FNAC is indicated when there is minimal sputum production or when tuberculosis presents as a mass lesion. Material for pulmonary tuberculosis is usually obtained by ultrasound or computed tomography (CT) guided FNAC. Other than FNAC, pleural fluid is also subjected for cytological examination in suspected case of pulmonary tuberculosis with pleural effusion. Proper planning is required as sample should be preserved for other investigations like ZiehlNeelsen (ZN) staining, culture and molecular techniques. Tuberculosis may show different types of cytomorphological patterns. These include (a) epithelioid cell granuloma with necrosis (Fig. 1), (b) only epithelioid cell granuloma without necrosis, (c) only necrotic material (Fig. 2), (d) 994
Diagnostic Cytopathology, Vol. 42, No 11
Fig. 3. Strong acid fast positivity. (Ziehl Neelsen stain 3 oil immersion magnification). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
rarely the cytology smears show only reactive lymphoid cells. AFB is frequently demonstrated in necrosis by Z-N staining (Fig. 3), (e) necrotic suppurative inflammation with moderate to numerous neutrophils lying in a watery necrotic background. Of all the above-mentioned cytological patterns, epithelioid cell granuloma with necrosis is the commonest combination. Epithelioid cells are modified histiocytes, which are elongated. They contain elongated carrot-shaped or slipper-shaped nuclei, straight or slightly curved. They are usually present in syncytium, loose clusters or singly. Small collections of epithelioid cells with or without surrounding lymphocytes are described as granulomas. Granulomas in tuberculosis may be well formed, compact or they may be vague, composed of loose clusters of epithelioid cells. Granulomas are variably accompanied by Langhans’ giant cells. These giant cells are large, 25–150 mm in size, contain multiple nuclei arranged peripherally. The aspirate in tuberculosis is whitish, cheesy, pus-like (caseous) necrotic material. Microscopically it shows necrotic material, which appears granular, eosinophilic, and usually devoid of nuclear debris (Fig. 2). These necrotic areas are usually accompanied by other inflammatory cells, predominantly epithelioid cells, macrophages, lymphocytes, and uncommonly neutrophils. Apart from these three common patterns, rarely only suppurative neutrophil rich inflammation is also found. This shows areas of necrosis accompanied by nuclear debris, collection of neurophils and variable number of histiocytes and lymphocytes. It might resemble acute necrotizing inflammation. The necrotic suppurative cytomorphologic features of tuberculosis is usually teeming with acid fast bacilii. Usually the gross appearance of the aspirated material is puslike and smears should be always prepared from purulent material for ZN staining. The overall AFB positivity ranges from 25 to 60% and depends on the type of
Diagnostic Cytopathology DOI 10.1002/dc
TUBERCULOSIS
aspirated material.8,9 The probability of AFB positivity is highest with only necrosis (around 70%), followed by epithelioid cell granuloma accompanied by necrosis (around 60%) and least with only epithelioid cell granuloma (around 20%).9 Pleural fluid samples are received in sodium oxalate preservative. They can be processed either by conventional technique like centrifugation or by liquid-based cytology (LBC) technique. Smears are prepared from the sediment. Pleural fluid in tuberculosis is usually lymphocyte rich, accompanied by few macrophages and mesothelial cells.10 Granulocytes are seen less frequently. Tuberculosis can also be responsible for eosinophilic pleural effusion.11,12 Occasionally it may lead to a plasma cell rich pleural effusion.13 Epithelioid cell granuloma or necrosis is extremely uncommon. Acid fast bacilli are only rarely found in pleural fluid and yield of mycobacterial culture is also poor. The possibility of a positive pleural fluid culture shows negative correlation with lymphocyte percentage in pleural fluid.14 Mesothelial cells are usually scanty in tuberculous pleural effusion compared with other reactive conditions, but their presence does not exclude the diagnosis of tuberculosis.15,16 Numerous mesothelial cells can be found in pleural fluid in HIV patients, possibly due to altered immune response.17 Direct pleural involvement by tuberculosis may cause marked reactive mesothelial proliferation in sheets and clusters. These may resemble mesothelioma or metastatic adenocarcinoma, and result in diagnostic dilemma.18 Additional markers like adenosine deaminase and interferon gamma level in pleural fluid are increased in tuberculosis. These tests suggest a possibility of tuberculosis, but are not diagnostic.19 Molecular techniques can improve diagnostic accuracy, which are discussed subsequently with ancillary techniques. Tracheal aspirates, bronchial washings, and bronchial brushings are also sometimes used for detection of endobronchial tuberculosis, especially when they mimic endobronchial growth. Tracheal aspirate slides should be directly submitted for AFB staining and culture. Tracheal brushing in tuberculosis usually show nonspecific inflammation composed of lymphocytes, macrophages, and few polymorphs.20 Epithelioid cell granuloma or necrosis is seen in 16–20% of cases. However, AFB positivity is usually higher (up to 40%). The total yield of bronchial washing in sputum negative pulmonary tuberculosis may be as high as 83%.21 These procedures are invasive, so they are not used in routine practice unless specifically indicated. Lymph node is the commonest extrapulmonary site of involvement in tuberculosis. Tuberculosis is responsible for 38–51% of clinically significant lymphadenitis in India.22,23 FNAC is considered as an easy and rapid method for the diagnosis of tuberculous lymphadenitis.9 Superficially located lymph nodes can be aspirated
directly using 23G needle. For deep seated lymph nodes like retroperitoneal or mediastinal lymph nodes, CT scan or ultrasound guided FNAC is employed. Transbronchialneedle aspiration (TBNA) is a useful method to sample subcarinal lymph nodes. FNAC from tuberculous lymphadenitis yields a similar cytomorphological pattern as in pulmonary tuberculosis.9,23 Following treatment with antituberculous drugs, most of the lymph nodes regress in size, some show reactive lymphoid hyperplasia with an excess of reactive plasma cells. In cases of good response to chemotherapy, there is intense lymphocytic infiltrate in the granulomas. In cases of recurrence, necrosis is the commonest pattern followed by granulomatous inflammation. However, frequency of AFB positivity and culture positivity usually reduces following antitubercular therapy.24 Another pattern of lymph node aspiration morphology has been described, which resembles mesenchymal tumors. The aspiration cytology is composed predominantly of spindle-shaped histiocytes with minimal necrosis and it mimics a spindle cell tumor. In setting of immunosuppression it can be misdiagnosed as Kaposi’s sarcoma or other sarcomas. This condition is called mycobacterial spindle cell pseudotumor.25 However, a careful search for epithelioid cells and AFB positivity can confirm the correct diagnosis. Apart from lung and lymph node, FNAC can be used to diagnose tuberculosis of virtually every organ including bone, soft tissue, pancreas, skin, thyroid, etc.26–31 Urine examination and cerebrospinal fluid (CSF) examination are also performed as a diagnostic work-up of genitourinary tuberculosis and tuberculous meningitis (TBM) respectively. Urinary tract tuberculosis is relatively common in developing countries. Patients may present with dysuria, burning sensation or hematuria, which may clinically mimic malignancy. Urinary cytology may reveal epitheloid cell granulomas, multinucleated giant cells associated with variable amount of necrosis.32 The granulomas are not usually very wellformed, rather loose clusters of epithelioid cells are seen. Positive stain for AFB can confirm the diagnosis. There may be reactive atypia of the urothelial cells as reported by Piscioli et al.33 It may resemble urothelial carcinoma in a given clinical setting. However, in an inflammatory background with epithelioid cells, mild to moderate cytological atypia is acceptable. However, granulomas in urine specimens are not specific for tuberculosis, but can be, it can be seen following BCG treatment for urinary bladder carcinoma or previous surgical intervention.34 In patients who have recieved local BCG treatment for carcinoma of the urinary bladder, follow up urine specimens with cytological atypia and granulomas may pose a significant diagnostic challenge A cystoscopic biopsy is mandatory in such cases to rule out a recurrence of tumor. Diagnostic Cytopathology, Vol. 42, No 11
995
Diagnostic Cytopathology DOI 10.1002/dc
CHATTERJEE AND DEY
CSF examination is an important diagnostic test for TBM. It includes both cytological, microbiological as well as biochemical evaluation. The classical CSF pattern described in TBM is lymphocytic pleocytosis. But this pattern is seen in 60–70% of TBM patients. In 20–30% of patients, a neutrophilic predominance may be seen.35 Thus a neutrophilic pleocytosis in CSF does not rule out the possibility of TBM, and in endemic countries, culture, and molecular diagnosis are required. Epithelioid cell granulomas in CSF are extremely rare finding. Bacteriological diagnosis, demonstration of acid fast bacilli (AFB) by Ziehl-Neelsen stain (sensitivity 4–40%) and culture (sensitivity 18–83%) is highly specific.36 Large number of bacteria (>10,000/mL) must be present to reliably detect AFB in smear. According to Indian reports, the yield of CSF culture is much lower. CSF collected by ventricular tap yields better result, although this is not practiced routinely. For a rapid and accurate diagnosis of TBM, molecular techniques involving nucleic acid amplification are the need of time (discussed later).
Tuberculosis in HIV-Positive Patients Tuberculosis is one of the commonest infections in retropositive cases. It is the major cause of death in AIDS patients and can be seen in up to 70% of these patients.2 FNAC is frequently employed for rapid diagnosis of tuberculosis in HIV positive patients as it is minimally invasive. In addition to the common patterns seen in immunecompetent patients, two additional morphological patterns can be seen in these patients.37,38 They include aspiration of acellular necrotic material and necrotic suppurative material. Another pattern seen in advanced disease is reactive lymphoid population with few macrophages, without any necrosis or granuloma (so called nonreactive tuberculosis).39 Nonreactive tuberculosis is seen in setting of immune suppression. The body is not able to mount an immune response. There may be large amount of necrosis or abscess formation. They show high bacillary load. ZN stain should be performed in all cases as acid fast bacilli can be seen even without a morphological suggestion of tuberculosis. AFB positivity is usually high.2,40 In a case of advanced HIV lymphadenopathy, lymph nodes are usually in atrophic, burnt out phase with a few lymphocytes, macrophages, plasma cells, fibrosis, and capillary proliferation.41 Dual infection and nontuberculous mycobacterial infection is common in HIV patients. Morphologically it is difficult to distinguish tuberculous bacilli from nontuberculous mycobacteria. Aspirated material should always be submitted for culture and molecular techniques.
Differential Diagnosis of Granulomatous Inflammation Apart from tuberculosis many other infectious and noninfectious conditions can produce granulomatous inflamma996
Diagnostic Cytopathology, Vol. 42, No 11
tion. In pulmonary tuberculosis, the major differential diagnoses include sarcoidosis, fungal granulomas and vasculitis like Wegener’s granulomatosis. The granulomas in sarcoidosis are usually compact and are not accompanied by necrosis.42 However, 25–30% sarcoid granulomas can show fibrinoid necrosis, which needs to be differentiated from caseous necrosis of tuberculosis in histopathology. Small group of scattered epithelioid cells, admixed with lymphocytes without multinucleated giant cells and necrosis may be more suggestive of sarcoidosis.43 Giant cells in sarcoidosis may be of Langhans’ type or foreign body type. The giant cells may contain different types of inclusions like Asteroid bodies, Schaumann bodies and Hamazaki-Wisenberg bodies. However, these features are difficult to appreciate in cytology. Stain for AFB is consistently negative. However, sarcoidosis cannot be differentiated from non-necrotic tuberculosis only by cytomorphology and other clinical and biochemical parameters should be taken into account.44 Fungal granulomas on the other hand are usually accompanied by necrosis and mixed inflammatory infiltrate composed of neutrophils, eosinophils, and lymphocytes. Fungal profiles are seen either on routine staining or by special stains like Periodic acid Schiff (PAS) or Grocott. Granulomatosis with polyarteritis (GPA, previously known as Wegener’s granulomatosis) is characterized by necrotizing granulomatous inflammation accompanied by vasculitis. Cytological examination reveals necrosis, variable inflammation, ill-formed epithelioid cell granulomas with or without giant cells. Atypical epithelioid cells may be present, which may mimic a malignant pathology.45 As both show necrotizing granulomas with variable inflammation, it may be extremely difficult to differentiate GPA from tuberculosis on cytological basis alone.46
Nontuberculous Mycobacteria (NTM) Infection NTM includes many mycobacterial species which can infect immune-compromised as well as immune-competent patients. One of the commonest manifestations is Buruli ulcer, a cutaneous infectious disease caused by Mycobacterium ulcerans. Other organs may be involved including lung, intestine, osteoarticular system, lymph node, soft tissue etc.47 More than 120 species of NTM have been recognized and 60% of them can cause human disease. NTM can cause fulminant infection in immune-compromised patients. Diagnosis of NTM is often difficult and challenging. FNAC has not been widely evaluated for the diagnosis of NTM infection. FNA may yield only necrotic material and inflammatory cells with or without necrosis. Aspiration from an immune-compromised host may reveal only collection of foamy macrophages. Stain for AFB is variable, it may be completely negative to strongly positive. Cases showing only necrosis or foamy macrophages are usually strongly positive for AFB. The sensitivity of AFB staining is 33–
Diagnostic Cytopathology DOI 10.1002/dc
TUBERCULOSIS
40% for diagnosis of Mycobacterium ulcerans infection.48 In HIV positive patients the aspirate may show many negative stained bacilli on the Romanowsky stains. They appear as cylindrical shaped or slit like negative images, which can be better appreciated by lowering the condenser of the microscope. This phenomenon occurs because of presence of lipid rich coat on cell wall of Mycobacteria which resist staining by the Romanowsky group of stains. These can present in extracellular spaces as well as within the cytoplasm of the foamy macrophages. Negative staining bacilli may be seen in aspiration cytology or bronchoalveolar lavage (BAL) fluid. This phenomenon is usually seen in Mycobacterium avium-intracelullare complex (MAC) infection in a setting of immune-suppression.49,50 Stain for AFB is usually strongly positive. Another important group of infection is disseminated Mycobacterium bovis infection after BCG vaccination. This is seen in children, usually less than 3 years of age.51 Children are routinely vaccinated with BCG shortly after birthfor the prevention of tuberculosis, in many countries. In children with defective cell mediate immunity, it can cause disseminated infection. The common conditions associated with disseminated BCG infection include severe combined immune-deficiency (SCID), chronic granulomatous disease, and HIV infection.52 BCG vaccination is given by intradermal injection. Local infection at the injection site and local lymphadenitis following BCG vaccination can be seen in both immunocompromised as well as immunocompetent children. Inravesical BCG therapy in case of urothelial carcinoma is usually a safe procedure. However, occasionally systemic BCG infection may occur in such cases. This may cause bronchopulmonary infection and granulomatous hepatitis. Morphologically NTM cannot be distinguished from Mycobacterium tuberculosis. To obtain a definite diagnosis of NTM infection, culture, and molecular techniques like PCR remain the method of choice. The sensitivity of nested PCR may be as high as 85%.53 FNAC can be used to monitor a patient on chemotherapy for NTM infection.
Tuberculosis and Malignancy The clinical manifestation of tuberculosis is highly variable and may even resemble malignancy. Pelvic tuberculosis on many occasions mimic an advanced ovarian carcinoma.54 Similarly pulmonary tuberculosis with central nervous system involvement may be confused as pulmonary carcinoma with systemic metastasis.55 In a suspected case of malignancy, presence of only necrotic material in FNAC without presence of malignant cells should raise the suspicion of tuberculosis. A simple ZN stain may help to achieve the correct diagnosis. However, the relationship between tuberculosis and malignancy is not well described. Tuberculosis and malignancy may coexist in a given patient in the same location.56 Among the malignancies, lung carcinoma and lymphomas have
been described to be associated with tuberculosis.57 Usually dual pathology is seen more commonly in the setting of immune-suppression, like HIV infection or organ transplant. Tumor itself and chemotherapy causes systemic immune-suppression which may be responsible for reactivation of tuberculosis. Coexistence of tuberculosis with lymphoma, both Hodgkin’s and Non Hodgkin’s lymphoma are well described in the literature.58,59 As both the conditions may present with lymphadenopathy and may clinically mimic one another, a correct tissue diagnosis is very important for institution of proper treatment. Lymphoma can produce a functional immunocompromized state. Both the conditions may produce necrosis, so a careful analysis of cell cytomorphology with special stains, flow cytometry and cell block followed by immunocytochemistry may help to arrive at the correct diagnosis. Tuberculosis can coexist with other malignancies in immunosuppressed conditions. There are reports of coexisting tuberculosis and Kaposi’s sarcoma in renal allograft recipients.60,61 Kaposi’s sarcoma can also present as lymph nodal enlargement. So in immunocompromized patients both primary or acquired, a possibility of double pathology should be kept in mind, especially if the patient does not respond to the treatment and a proper cytological evaluation supplemented by ancillary techniques may guide the clinician to institute a proper treatment.
Ancillary Diagnostic Techniques Although the tissue reaction in tuberculosis is quite suggestive, an accurate diagnosis is often required as other conditions including various infections and malignancies may sometime produce similar morphological changes. It is therefore required to demonstrate the organism either by ZN staining or by culture. The sensitivity of detection of AFB in cytological material is variable in different studies, overall ranges from 40 to 78%. The incidence of AFB positivity also depends on the cytomorphology.8,9 Culture from FNA material shows variable sensitivity, varying from 32 to 35%. The sensitivity of culture also varies with the nature of the aspirated material. Mycobacteria are isolated more frequently from caseating lesions than noncaseating lesions.62,63 Mycobacterial culture is extremely specific, but the disadvantages are poor sensitivity and the prolonged time taken for culture. Culture in Lowenstein-Jensen media requires 8–12 weeks for isolation of the bacteria. BACTEC culture takes significantly less time (average 3 weeks) with slightly improved sensitivity.64 Bactec MGIT960 liquid medium used worldwide as a medium for mycobacterial culture with excellent results. Compared with conventional LJ medium, it shows higher sensitivity for detection of tuberculous bacilli with a much lower mean time for detection (average 10–18 days for MGIT 960 and average 30–50 days in LJ media).65 Fluorescent microscopy with auramineDiagnostic Cytopathology, Vol. 42, No 11
997
Diagnostic Cytopathology DOI 10.1002/dc
CHATTERJEE AND DEY
Fig. 4. Multiplex PCR showing L2-positive control and L3 negative control along with test samples. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
rhodamine staining is also employed for demonstration of tuberculous bacilli on FNA material. The sensitivity is similar or slightly higher than conventional ZN staining. However, in the auramine–rhodamine procedure, the cytomorphology is distorted and the smear becomes useless for future reference. Moreover, it is expensive, involves complex technique and requires fluorescent microscope for interpretation.66 So this method is not very popular in routine practice. Mycobacteria also show autofluorescence, as seen on the Papanicolaou stain. This is a simple procedure and does not require any special fluorescent dye, so considered quite useful in resource poor settings. It can be applied directly to Papanicolaou-stained smears. Auto-fluorescence is done under invisible ultraviolet light, using wavelength of 350–400 micron. This method markedly increases the sensitivity of detection of mycobacterial over conventional ZN stain, with sensitivity as high as 95%.67 This can detect bacilli when present in low concentration (
Tuberculosis is a major public health problem, especially in developing countries. The clinical manifestations of tuberculosis are very nonspecific and may mimic many other conditions. An accurate diagnosis is extremely important as the disease is treatable with antituberculous therapy. Cytological examination can provide useful diagnostic material for routine and ancillary techniques for rapid and accurate diagnosis of tuberculosis. Here we review the different morphological patterns of tuberculosis, challenges, and application of ancillary techniques for cytological diagnosis of tuberculosis. Diagn. Cytopathol. 2014;42:993–1001. VC 2014 Wiley Periodicals, Inc. Key Words:
tuberculosis; cytology; FNAC; lymph node; PCR
Introduction Tuberculosis is a major public health problem, especially in developing countries. According to the WHO Global tuberculosis report of 2012, the overall estimated global incidence of tuberculosis is 8.7 million, half of which is shared by India and China.1 In the western world, there is again a resurgence of tuberculosis because of HIV coinfection. Tuberculosis remains the major cause of death among AIDS patients, with up to 29% showing central nervous system (CNS) involvement.2 Pulmonary tuberculosis is the commonest form of disease. Among the extra-pulmonary locations, tuberculous lymphadenitis is the most frequent. Central nervous system involvement is seen around 10% of tuberculosis patients and represents the most serious legacy.3 Tuberculosis is caused by Mycobacterium tuberculosis, which is an aerobic, nonmotile and noncapsulated bacterium. Mycobacteria resist decolourisation by dilute
1 Department of Pathology, Post Graduate Institute of Medical Education and Research, PGIMER, Chandigarh, India 2 Department of Cytology, Post Graduate Institute of Medical Education and Research, Chandigarh, India *Correspondence to: Dr Pranab Dey, MD, MIAC, FRCPath, Department of Cytology, Postgraduate Institute of Medical Education and Research, Chandigarh, India. E-mail: [email protected] Received 30 December 2013; Accepted 11 June 2014 DOI: 10.1002/dc.23190 Published online 27 June 2014 in Wiley Online Library (wileyonlinelibrary.com).
C 2014 WILEY PERIODICALS, INC. V
mineral acid; therefore, mycobacteria are called “acid fast bacilli” or AFB. The term M. tuberculosis complex includes M. tuberculosis, M. bovis, M. africanum, and M. microti. M. microti does not cause human disease.4 Atypical mycobacteria, also known as “nontuberculous mycobacteria” (NTM) cause opportunistic infections in immunocompromized hosts. They include M. avium, M. intracellulare, M. kansasii, M. marinum, etc. Morphologically they cannot be distinguished from M. tuberculosis. This distinction is important as treatment of NTM infection is different from tuberculosis.5 The clinical manifestations of tuberculosis are very nonspecific and may mimic many other conditions. An accurate diagnosis is extremely important as the disease is treatable with antitubercular therapy. Cytological examination can provide useful diagnostic material for routine practice and ancillary techniques for rapid and accurate diagnosis of tuberculosis. Here we review the different morphological pattern of tuberculosis, challenges, and application of ancillary techniques for cytological diagnosis of tuberculosis.
Clinical Manifestations: Pulmonary Tuberculosis Lung is the primary site of infection by M. tuberculosis. Pulmonary tuberculosis may be primary or post-primary (secondary). Primary tuberculosis and progressive primary tuberculosis is seen mainly in children. Post primary tuberculosis is usually a disease of adult life. Primary tuberculosis is characterized by granulomatous inflammation. The main pathology of post primary tuberculosis is lipoid pneumonia, followed by caseous pneumonia and fibrocavitary lesion.6 Pulmonary tuberculosis is usually diagnosed by sputum microscopy for Acid fast bacilli (AFB), accompanied by culture or molecular techniques. The role of fine-needle aspiration cytology (FNAC) is limited in the diagnosis of pulmonary tuberculosis. Sputum examination for AFB is an integral part of global TB control strategy, and usually done on 3 consecutive days. Usually sputum screening for AFB is not done by cytologists. Sputum microscopy is a very cost effective and simple test but lacks sensitivity, especially when the bacterial concentration in sputum Diagnostic Cytopathology, Vol. 42, No 11
993
Diagnostic Cytopathology DOI 10.1002/dc
CHATTERJEE AND DEY
Fig. 1. Epithelioid cell granulomas along with large number of inflammatory cells. (May Grunwald Giemsa 3 higher magnification). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary. com.]
Fig. 2. Necrosis in tuberculosis (May Grunwald Giemsa 3 higher magnification). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
is less than 10,000/mL. The sensitivity can be increased by use of fluorescent microscopy.7 For children where sputum is difficult to obtain, gastric lavage for AFB is used as a screening modality for tuberculosis. FNAC is indicated when there is minimal sputum production or when tuberculosis presents as a mass lesion. Material for pulmonary tuberculosis is usually obtained by ultrasound or computed tomography (CT) guided FNAC. Other than FNAC, pleural fluid is also subjected for cytological examination in suspected case of pulmonary tuberculosis with pleural effusion. Proper planning is required as sample should be preserved for other investigations like ZiehlNeelsen (ZN) staining, culture and molecular techniques. Tuberculosis may show different types of cytomorphological patterns. These include (a) epithelioid cell granuloma with necrosis (Fig. 1), (b) only epithelioid cell granuloma without necrosis, (c) only necrotic material (Fig. 2), (d) 994
Diagnostic Cytopathology, Vol. 42, No 11
Fig. 3. Strong acid fast positivity. (Ziehl Neelsen stain 3 oil immersion magnification). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
rarely the cytology smears show only reactive lymphoid cells. AFB is frequently demonstrated in necrosis by Z-N staining (Fig. 3), (e) necrotic suppurative inflammation with moderate to numerous neutrophils lying in a watery necrotic background. Of all the above-mentioned cytological patterns, epithelioid cell granuloma with necrosis is the commonest combination. Epithelioid cells are modified histiocytes, which are elongated. They contain elongated carrot-shaped or slipper-shaped nuclei, straight or slightly curved. They are usually present in syncytium, loose clusters or singly. Small collections of epithelioid cells with or without surrounding lymphocytes are described as granulomas. Granulomas in tuberculosis may be well formed, compact or they may be vague, composed of loose clusters of epithelioid cells. Granulomas are variably accompanied by Langhans’ giant cells. These giant cells are large, 25–150 mm in size, contain multiple nuclei arranged peripherally. The aspirate in tuberculosis is whitish, cheesy, pus-like (caseous) necrotic material. Microscopically it shows necrotic material, which appears granular, eosinophilic, and usually devoid of nuclear debris (Fig. 2). These necrotic areas are usually accompanied by other inflammatory cells, predominantly epithelioid cells, macrophages, lymphocytes, and uncommonly neutrophils. Apart from these three common patterns, rarely only suppurative neutrophil rich inflammation is also found. This shows areas of necrosis accompanied by nuclear debris, collection of neurophils and variable number of histiocytes and lymphocytes. It might resemble acute necrotizing inflammation. The necrotic suppurative cytomorphologic features of tuberculosis is usually teeming with acid fast bacilii. Usually the gross appearance of the aspirated material is puslike and smears should be always prepared from purulent material for ZN staining. The overall AFB positivity ranges from 25 to 60% and depends on the type of
Diagnostic Cytopathology DOI 10.1002/dc
TUBERCULOSIS
aspirated material.8,9 The probability of AFB positivity is highest with only necrosis (around 70%), followed by epithelioid cell granuloma accompanied by necrosis (around 60%) and least with only epithelioid cell granuloma (around 20%).9 Pleural fluid samples are received in sodium oxalate preservative. They can be processed either by conventional technique like centrifugation or by liquid-based cytology (LBC) technique. Smears are prepared from the sediment. Pleural fluid in tuberculosis is usually lymphocyte rich, accompanied by few macrophages and mesothelial cells.10 Granulocytes are seen less frequently. Tuberculosis can also be responsible for eosinophilic pleural effusion.11,12 Occasionally it may lead to a plasma cell rich pleural effusion.13 Epithelioid cell granuloma or necrosis is extremely uncommon. Acid fast bacilli are only rarely found in pleural fluid and yield of mycobacterial culture is also poor. The possibility of a positive pleural fluid culture shows negative correlation with lymphocyte percentage in pleural fluid.14 Mesothelial cells are usually scanty in tuberculous pleural effusion compared with other reactive conditions, but their presence does not exclude the diagnosis of tuberculosis.15,16 Numerous mesothelial cells can be found in pleural fluid in HIV patients, possibly due to altered immune response.17 Direct pleural involvement by tuberculosis may cause marked reactive mesothelial proliferation in sheets and clusters. These may resemble mesothelioma or metastatic adenocarcinoma, and result in diagnostic dilemma.18 Additional markers like adenosine deaminase and interferon gamma level in pleural fluid are increased in tuberculosis. These tests suggest a possibility of tuberculosis, but are not diagnostic.19 Molecular techniques can improve diagnostic accuracy, which are discussed subsequently with ancillary techniques. Tracheal aspirates, bronchial washings, and bronchial brushings are also sometimes used for detection of endobronchial tuberculosis, especially when they mimic endobronchial growth. Tracheal aspirate slides should be directly submitted for AFB staining and culture. Tracheal brushing in tuberculosis usually show nonspecific inflammation composed of lymphocytes, macrophages, and few polymorphs.20 Epithelioid cell granuloma or necrosis is seen in 16–20% of cases. However, AFB positivity is usually higher (up to 40%). The total yield of bronchial washing in sputum negative pulmonary tuberculosis may be as high as 83%.21 These procedures are invasive, so they are not used in routine practice unless specifically indicated. Lymph node is the commonest extrapulmonary site of involvement in tuberculosis. Tuberculosis is responsible for 38–51% of clinically significant lymphadenitis in India.22,23 FNAC is considered as an easy and rapid method for the diagnosis of tuberculous lymphadenitis.9 Superficially located lymph nodes can be aspirated
directly using 23G needle. For deep seated lymph nodes like retroperitoneal or mediastinal lymph nodes, CT scan or ultrasound guided FNAC is employed. Transbronchialneedle aspiration (TBNA) is a useful method to sample subcarinal lymph nodes. FNAC from tuberculous lymphadenitis yields a similar cytomorphological pattern as in pulmonary tuberculosis.9,23 Following treatment with antituberculous drugs, most of the lymph nodes regress in size, some show reactive lymphoid hyperplasia with an excess of reactive plasma cells. In cases of good response to chemotherapy, there is intense lymphocytic infiltrate in the granulomas. In cases of recurrence, necrosis is the commonest pattern followed by granulomatous inflammation. However, frequency of AFB positivity and culture positivity usually reduces following antitubercular therapy.24 Another pattern of lymph node aspiration morphology has been described, which resembles mesenchymal tumors. The aspiration cytology is composed predominantly of spindle-shaped histiocytes with minimal necrosis and it mimics a spindle cell tumor. In setting of immunosuppression it can be misdiagnosed as Kaposi’s sarcoma or other sarcomas. This condition is called mycobacterial spindle cell pseudotumor.25 However, a careful search for epithelioid cells and AFB positivity can confirm the correct diagnosis. Apart from lung and lymph node, FNAC can be used to diagnose tuberculosis of virtually every organ including bone, soft tissue, pancreas, skin, thyroid, etc.26–31 Urine examination and cerebrospinal fluid (CSF) examination are also performed as a diagnostic work-up of genitourinary tuberculosis and tuberculous meningitis (TBM) respectively. Urinary tract tuberculosis is relatively common in developing countries. Patients may present with dysuria, burning sensation or hematuria, which may clinically mimic malignancy. Urinary cytology may reveal epitheloid cell granulomas, multinucleated giant cells associated with variable amount of necrosis.32 The granulomas are not usually very wellformed, rather loose clusters of epithelioid cells are seen. Positive stain for AFB can confirm the diagnosis. There may be reactive atypia of the urothelial cells as reported by Piscioli et al.33 It may resemble urothelial carcinoma in a given clinical setting. However, in an inflammatory background with epithelioid cells, mild to moderate cytological atypia is acceptable. However, granulomas in urine specimens are not specific for tuberculosis, but can be, it can be seen following BCG treatment for urinary bladder carcinoma or previous surgical intervention.34 In patients who have recieved local BCG treatment for carcinoma of the urinary bladder, follow up urine specimens with cytological atypia and granulomas may pose a significant diagnostic challenge A cystoscopic biopsy is mandatory in such cases to rule out a recurrence of tumor. Diagnostic Cytopathology, Vol. 42, No 11
995
Diagnostic Cytopathology DOI 10.1002/dc
CHATTERJEE AND DEY
CSF examination is an important diagnostic test for TBM. It includes both cytological, microbiological as well as biochemical evaluation. The classical CSF pattern described in TBM is lymphocytic pleocytosis. But this pattern is seen in 60–70% of TBM patients. In 20–30% of patients, a neutrophilic predominance may be seen.35 Thus a neutrophilic pleocytosis in CSF does not rule out the possibility of TBM, and in endemic countries, culture, and molecular diagnosis are required. Epithelioid cell granulomas in CSF are extremely rare finding. Bacteriological diagnosis, demonstration of acid fast bacilli (AFB) by Ziehl-Neelsen stain (sensitivity 4–40%) and culture (sensitivity 18–83%) is highly specific.36 Large number of bacteria (>10,000/mL) must be present to reliably detect AFB in smear. According to Indian reports, the yield of CSF culture is much lower. CSF collected by ventricular tap yields better result, although this is not practiced routinely. For a rapid and accurate diagnosis of TBM, molecular techniques involving nucleic acid amplification are the need of time (discussed later).
Tuberculosis in HIV-Positive Patients Tuberculosis is one of the commonest infections in retropositive cases. It is the major cause of death in AIDS patients and can be seen in up to 70% of these patients.2 FNAC is frequently employed for rapid diagnosis of tuberculosis in HIV positive patients as it is minimally invasive. In addition to the common patterns seen in immunecompetent patients, two additional morphological patterns can be seen in these patients.37,38 They include aspiration of acellular necrotic material and necrotic suppurative material. Another pattern seen in advanced disease is reactive lymphoid population with few macrophages, without any necrosis or granuloma (so called nonreactive tuberculosis).39 Nonreactive tuberculosis is seen in setting of immune suppression. The body is not able to mount an immune response. There may be large amount of necrosis or abscess formation. They show high bacillary load. ZN stain should be performed in all cases as acid fast bacilli can be seen even without a morphological suggestion of tuberculosis. AFB positivity is usually high.2,40 In a case of advanced HIV lymphadenopathy, lymph nodes are usually in atrophic, burnt out phase with a few lymphocytes, macrophages, plasma cells, fibrosis, and capillary proliferation.41 Dual infection and nontuberculous mycobacterial infection is common in HIV patients. Morphologically it is difficult to distinguish tuberculous bacilli from nontuberculous mycobacteria. Aspirated material should always be submitted for culture and molecular techniques.
Differential Diagnosis of Granulomatous Inflammation Apart from tuberculosis many other infectious and noninfectious conditions can produce granulomatous inflamma996
Diagnostic Cytopathology, Vol. 42, No 11
tion. In pulmonary tuberculosis, the major differential diagnoses include sarcoidosis, fungal granulomas and vasculitis like Wegener’s granulomatosis. The granulomas in sarcoidosis are usually compact and are not accompanied by necrosis.42 However, 25–30% sarcoid granulomas can show fibrinoid necrosis, which needs to be differentiated from caseous necrosis of tuberculosis in histopathology. Small group of scattered epithelioid cells, admixed with lymphocytes without multinucleated giant cells and necrosis may be more suggestive of sarcoidosis.43 Giant cells in sarcoidosis may be of Langhans’ type or foreign body type. The giant cells may contain different types of inclusions like Asteroid bodies, Schaumann bodies and Hamazaki-Wisenberg bodies. However, these features are difficult to appreciate in cytology. Stain for AFB is consistently negative. However, sarcoidosis cannot be differentiated from non-necrotic tuberculosis only by cytomorphology and other clinical and biochemical parameters should be taken into account.44 Fungal granulomas on the other hand are usually accompanied by necrosis and mixed inflammatory infiltrate composed of neutrophils, eosinophils, and lymphocytes. Fungal profiles are seen either on routine staining or by special stains like Periodic acid Schiff (PAS) or Grocott. Granulomatosis with polyarteritis (GPA, previously known as Wegener’s granulomatosis) is characterized by necrotizing granulomatous inflammation accompanied by vasculitis. Cytological examination reveals necrosis, variable inflammation, ill-formed epithelioid cell granulomas with or without giant cells. Atypical epithelioid cells may be present, which may mimic a malignant pathology.45 As both show necrotizing granulomas with variable inflammation, it may be extremely difficult to differentiate GPA from tuberculosis on cytological basis alone.46
Nontuberculous Mycobacteria (NTM) Infection NTM includes many mycobacterial species which can infect immune-compromised as well as immune-competent patients. One of the commonest manifestations is Buruli ulcer, a cutaneous infectious disease caused by Mycobacterium ulcerans. Other organs may be involved including lung, intestine, osteoarticular system, lymph node, soft tissue etc.47 More than 120 species of NTM have been recognized and 60% of them can cause human disease. NTM can cause fulminant infection in immune-compromised patients. Diagnosis of NTM is often difficult and challenging. FNAC has not been widely evaluated for the diagnosis of NTM infection. FNA may yield only necrotic material and inflammatory cells with or without necrosis. Aspiration from an immune-compromised host may reveal only collection of foamy macrophages. Stain for AFB is variable, it may be completely negative to strongly positive. Cases showing only necrosis or foamy macrophages are usually strongly positive for AFB. The sensitivity of AFB staining is 33–
Diagnostic Cytopathology DOI 10.1002/dc
TUBERCULOSIS
40% for diagnosis of Mycobacterium ulcerans infection.48 In HIV positive patients the aspirate may show many negative stained bacilli on the Romanowsky stains. They appear as cylindrical shaped or slit like negative images, which can be better appreciated by lowering the condenser of the microscope. This phenomenon occurs because of presence of lipid rich coat on cell wall of Mycobacteria which resist staining by the Romanowsky group of stains. These can present in extracellular spaces as well as within the cytoplasm of the foamy macrophages. Negative staining bacilli may be seen in aspiration cytology or bronchoalveolar lavage (BAL) fluid. This phenomenon is usually seen in Mycobacterium avium-intracelullare complex (MAC) infection in a setting of immune-suppression.49,50 Stain for AFB is usually strongly positive. Another important group of infection is disseminated Mycobacterium bovis infection after BCG vaccination. This is seen in children, usually less than 3 years of age.51 Children are routinely vaccinated with BCG shortly after birthfor the prevention of tuberculosis, in many countries. In children with defective cell mediate immunity, it can cause disseminated infection. The common conditions associated with disseminated BCG infection include severe combined immune-deficiency (SCID), chronic granulomatous disease, and HIV infection.52 BCG vaccination is given by intradermal injection. Local infection at the injection site and local lymphadenitis following BCG vaccination can be seen in both immunocompromised as well as immunocompetent children. Inravesical BCG therapy in case of urothelial carcinoma is usually a safe procedure. However, occasionally systemic BCG infection may occur in such cases. This may cause bronchopulmonary infection and granulomatous hepatitis. Morphologically NTM cannot be distinguished from Mycobacterium tuberculosis. To obtain a definite diagnosis of NTM infection, culture, and molecular techniques like PCR remain the method of choice. The sensitivity of nested PCR may be as high as 85%.53 FNAC can be used to monitor a patient on chemotherapy for NTM infection.
Tuberculosis and Malignancy The clinical manifestation of tuberculosis is highly variable and may even resemble malignancy. Pelvic tuberculosis on many occasions mimic an advanced ovarian carcinoma.54 Similarly pulmonary tuberculosis with central nervous system involvement may be confused as pulmonary carcinoma with systemic metastasis.55 In a suspected case of malignancy, presence of only necrotic material in FNAC without presence of malignant cells should raise the suspicion of tuberculosis. A simple ZN stain may help to achieve the correct diagnosis. However, the relationship between tuberculosis and malignancy is not well described. Tuberculosis and malignancy may coexist in a given patient in the same location.56 Among the malignancies, lung carcinoma and lymphomas have
been described to be associated with tuberculosis.57 Usually dual pathology is seen more commonly in the setting of immune-suppression, like HIV infection or organ transplant. Tumor itself and chemotherapy causes systemic immune-suppression which may be responsible for reactivation of tuberculosis. Coexistence of tuberculosis with lymphoma, both Hodgkin’s and Non Hodgkin’s lymphoma are well described in the literature.58,59 As both the conditions may present with lymphadenopathy and may clinically mimic one another, a correct tissue diagnosis is very important for institution of proper treatment. Lymphoma can produce a functional immunocompromized state. Both the conditions may produce necrosis, so a careful analysis of cell cytomorphology with special stains, flow cytometry and cell block followed by immunocytochemistry may help to arrive at the correct diagnosis. Tuberculosis can coexist with other malignancies in immunosuppressed conditions. There are reports of coexisting tuberculosis and Kaposi’s sarcoma in renal allograft recipients.60,61 Kaposi’s sarcoma can also present as lymph nodal enlargement. So in immunocompromized patients both primary or acquired, a possibility of double pathology should be kept in mind, especially if the patient does not respond to the treatment and a proper cytological evaluation supplemented by ancillary techniques may guide the clinician to institute a proper treatment.
Ancillary Diagnostic Techniques Although the tissue reaction in tuberculosis is quite suggestive, an accurate diagnosis is often required as other conditions including various infections and malignancies may sometime produce similar morphological changes. It is therefore required to demonstrate the organism either by ZN staining or by culture. The sensitivity of detection of AFB in cytological material is variable in different studies, overall ranges from 40 to 78%. The incidence of AFB positivity also depends on the cytomorphology.8,9 Culture from FNA material shows variable sensitivity, varying from 32 to 35%. The sensitivity of culture also varies with the nature of the aspirated material. Mycobacteria are isolated more frequently from caseating lesions than noncaseating lesions.62,63 Mycobacterial culture is extremely specific, but the disadvantages are poor sensitivity and the prolonged time taken for culture. Culture in Lowenstein-Jensen media requires 8–12 weeks for isolation of the bacteria. BACTEC culture takes significantly less time (average 3 weeks) with slightly improved sensitivity.64 Bactec MGIT960 liquid medium used worldwide as a medium for mycobacterial culture with excellent results. Compared with conventional LJ medium, it shows higher sensitivity for detection of tuberculous bacilli with a much lower mean time for detection (average 10–18 days for MGIT 960 and average 30–50 days in LJ media).65 Fluorescent microscopy with auramineDiagnostic Cytopathology, Vol. 42, No 11
997
Diagnostic Cytopathology DOI 10.1002/dc
CHATTERJEE AND DEY
Fig. 4. Multiplex PCR showing L2-positive control and L3 negative control along with test samples. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
rhodamine staining is also employed for demonstration of tuberculous bacilli on FNA material. The sensitivity is similar or slightly higher than conventional ZN staining. However, in the auramine–rhodamine procedure, the cytomorphology is distorted and the smear becomes useless for future reference. Moreover, it is expensive, involves complex technique and requires fluorescent microscope for interpretation.66 So this method is not very popular in routine practice. Mycobacteria also show autofluorescence, as seen on the Papanicolaou stain. This is a simple procedure and does not require any special fluorescent dye, so considered quite useful in resource poor settings. It can be applied directly to Papanicolaou-stained smears. Auto-fluorescence is done under invisible ultraviolet light, using wavelength of 350–400 micron. This method markedly increases the sensitivity of detection of mycobacterial over conventional ZN stain, with sensitivity as high as 95%.67 This can detect bacilli when present in low concentration (
