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Nucleic Acid Amplification of Mycobacterium tuberculosis Complex DNA from Archival Fine Needle Aspiration Smear Scrapings vs. Fresh Fine Needle Aspirates of Tuberculous Lymphadenitis

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Nucleic Acid Amplification of Mycobacterium tuberculosis Complex DNA from Archival Fine Needle Aspiration Smear Scrapings vs. Fresh Fine Needle Aspirates of Tuberculous Lymphadenitis

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Abstract

To assess the efficacy of the nucleic acid amplification (NAA) technique for Mycobacterium tuberculosis (MTB) complex from archival fine needle aspirate (FNA) smear scrapings of confirmed cases of extrapulmonary tuberculosis (EPTB) for a retrospective diagnosis of EPTB as compared to NAA from fresh FNA material from the same cases. Smear scrapings from 51 cases; 33 cases of tuberculous lymphadenitis (from patients who had undergone NAA 1 year before for MTB from fresh FNA material); 13 negative controls from nontuberculous, archival FNA smears; and 5 known acid-fast bacilli (AFB)-positive sputum smears, were subjected to NAA using the IS6110 primer sequence of M tuberculosis. Ziehl-Neelsen staining was done in all the smears. Of the 33 cases of tuberculous lymphadenitis, 15 (45.4%) were AFB positive and 18 (64.5%) AFB negative. MTB NAA was positive in 73.3% (11 of 15 AFB-positive cases) in the freshly aspirated material and was observed in 60% (9 of 15 AFB-positive cases) when done on DNA extracted from the archival smear scrapings of the same cases. Similarly, in the 18 AFB-negative cases, MTB NAA positivity was 72.2% (13 of 18) on fresh material and 44.4% (8 of 18) on archival smear scrapings from the same AFB-negative cases. Overall NAA positivity was 51.5% for archival smear scrapings as compared to 71% for fresh FNA of the same cases. Low NAA sensitivity of MTB DNA in archival material of known tuberculous cases limits the routine use of NAA based retrospective molecular diagnosis of MTB complex.

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... 18 These are not diagnosed by FNAC alone and subsequently are confirmed by other supportive tests. 19 Other causes of granulomatous thyroiditis are also to be ruled out. ZN staining has low detection sensitivity (0-75%) for AFB. ...
... ZN staining has low detection sensitivity (0-75%) for AFB. 18,19 An inverse relationship has been observed between granulomas and the presence of AFB. Purulent aspirates show the highest number of AFB and lowest number of granulomas, whereas blood-mixed aspirates show the highest number of granulomas and lowest number of bacilli in ZN staining. ...
... Otherwise the confirmation has to be done by histopathology of resected tissue or by a molecular diagnostic method such as polymerase chain reaction (PCR). [18][19][20] Molecular diagnostic methods for tuberculosis, 21 however, are still not available to routine laboratories in developing countries where tuberculosis is rampant. In developing countries, PCR is being performed in-house, without regulatory control. ...
Article
Tuberculosis of the thyroid is very rare and does not strike the clinician as a first clinical diagnosis of a thyroid nodule. To our knowledge, only 40 cases of tuberculous thyroiditis diagnosed by fine needle aspiration cytology (FNAC) are described in the English literature. We report a case of tuberculous thyroiditis in a young woman who presented with a right-side solitary thyroid nodule of short duration (15 days), diagnosed by FNAC and confirmed by positive immunocytochemistry with monoclonal antibody to Mycobacterium tuberculosis complex. Ziehl Neelsen staining for acid-fast bacilli (AFB) was negative. FNAC provides a confident preoperative diagnosis of thyroid tuberculosis, obviating the need for unnecessary surgical removal of thyroid nodule. Immunocytochemistry is an important diagnostic adjunct to FNAC in AFB-negative cases.
... Moreover they are still out of reach in routine diagnosis in resource poor countries. [1,2] Of the two forms of tuberculosis, EPTB is often difficult to Immunocytochemistry versus nucleic acid amplification in fine needle aspirates and tissues of extrapulmonary tuberculosis Goel, et al.: Immunocytochemistry and nucleic acid amplification in extrapulmonary tuberculosis diagnose because of its diverse clinical presentation and uncertain clinical evaluation. Lymph node is the most common type of EPTB and fine needle aspiration cytology (FNAC) is the first investigation advised. ...
... The diverse cytomorphological spectra [Plate 1] were noted in cytological smears of suspected cases of tuberculosis which were categorized in to seven groups. [1,3,4] Histological diagnosis of granulomatous lesions was made according to diagnostic criteria described in standard text. ...
... In house NAA for MTB complex DNA from fresh FNA material, archival and fresh FNA smears and tissue sections was done in three steps as standardized [1] in our laboratory: 1. Mycobacterial DNA extraction by phenol: chloroform method. 2. MTB complex DNA amplification by using IS6110 primer sequence. ...
Article
Full-text available
Immunocytochemistry (ICC) is an established routine diagnostic adjunct to cytology and histology for tumor diagnosis but has received little attention for diagnosis of tuberculosis. To have an objective method of direct visualization of mycobacteria or their products in clinical extrapulmonary tuberculosis (EPTB) specimens, immunocytochemical localization of M. tuberculosis antigen by staining with species specific monoclonal antibody to 38-kDa antigen of Mycobacterium tuberculosis complex. Immunostaining with specific monoclonal antibody to 38-kDa antigen of Mycobacterium tuberculosis complex was done in fresh and archival fine needle aspirates and tissue granulomata of 302 cases of extrapulmonary tuberculosis and was compared with the molecular diagnostic i.e., nucleic amplification and conventional [Cytomorphology, Ziehl Neelsen (ZN) staining and culture] tests and 386 controls. Diagnostic indices by Bayesian analysis for all types of archival and fresh material varied from 64 to 76% in nucleic acid amplification (NAA) and 96 to 98% in ICC. There was no significant difference in the diagnostic indices of ZN staining and/ or ICC in fresh or archival material whereas the sensitivity of NAA differed significantly in fresh versus archival material both in cytology (71.4% vs 52.1%) and histology (51.1% vs 38.8%). ICC can be easily used on archival smears and formalin-fixed paraffin-embedded tissue sections with almost equal sensitivity and specificity as with fresh material, in contrast to NAA which showed significant difference in test results on archival and fresh material. Low detection sensitivity of MTB DNA in archival material from known tuberculous cases showed the limitation of in-house NAA-based molecular diagnosis. ICC was found to be sensitive, specific and a better technique than NAA and can be used as an adjunct to conventional morphology and ZN staining for the diagnosis of EPTB in tissue granulomas.
... 33 Culture is regarded as the "gold standard" but needs 6-12 weeks and should be performed in a biosafety level 3 faculty. 34 Unfortunately, time is the most important factor in the diagnosis of an ID, particularly in immunocompromised conditions such as AIDS. In the study of Goel et al. nucleic acid testing (NAA) positivity for mycobacterial infection was reported in 72%-73% in fresh aspirates, regardless of the presence or absence of AFB. ...
... In the study of Goel et al. nucleic acid testing (NAA) positivity for mycobacterial infection was reported in 72%-73% in fresh aspirates, regardless of the presence or absence of AFB. 34 Based on the study of Pruhit et al. PCR provided high sensitivity, specificity, and positive and negative predictive values (85%, 95%, 96%, and 59%, respectively) by using unstained air-dried cytology smears in 98 cases for the early and specific diagnosis of EPTBC. ...
... 33 Culture is regarded as the "gold standard" but needs 6-12 weeks and should be performed in a biosafety level 3 faculty. 34 Unfortunately, time is the most important factor in the diagnosis of an ID, particularly in immunocompromised conditions such as AIDS. In the study of Goel et al. nucleic acid testing (NAA) positivity for mycobacterial infection was reported in 72%-73% in fresh aspirates, regardless of the presence or absence of AFB. ...
... In the study of Goel et al. nucleic acid testing (NAA) positivity for mycobacterial infection was reported in 72%-73% in fresh aspirates, regardless of the presence or absence of AFB. 34 Based on the study of Pruhit et al. PCR provided high sensitivity, specificity, and positive and negative predictive values (85%, 95%, 96%, and 59%, respectively) by using unstained air-dried cytology smears in 98 cases for the early and specific diagnosis of EPTBC. ...
Article
Full-text available
Pathologists have an important role in the diagnosis of infectious disease (ID). In many cases, a definitive diagnosis can be made using cytopathology alone. However, several ancillary techniques can be used on cytological material to reach a specific diagnosis by identifying the causative agent and consequently defining the management of the patient. This review aims to present the effectiveness of the application of molecular studies on cytological material to diagnose IDs and discuss the advantages and disadvantages of the various molecular techniques according to the type of cytological specimen and the infectious agents. Diagn. Cytopathol. 2015. © 2015 Wiley Periodicals, Inc.
... FNA also allows the performance of histochemical, immunohistochemical, and molecular techniques for the identification of infectious agents and the searching for oncogenic proteins and specific molecular changes causing disease. The results of FNA reported in the literature are comparable to the results of biopsy, [11][12][13][14] and for the diagnosis of a mycobacterial disease other than leprosy, it has been shown to be as specific as PCR. 15 Despite its use in diagnostic procedures, there are no reports of the use of FNA to collect material for the experimental inoculation of animals. ...
... It is also possible to perform several different studies on the FNA-collected material, with results comparable to studies conducted on biopsy material. [11][12][13][14][15]23,24 In both experimental models, for leprosy and Jorge Lobo's disease, the etiologic agents are inoculated in mice footpads. For passage into other animals or for collection of material to be studied, it is necessary to sacrifice the animal host to obtain enough bacilli or fungal cells. ...
Article
Full-text available
Procedures involving the use of Mycobacterium leprae and Lacazia loboi, uncultivated organisms, depend on the collection of material from the lesions of patients or experimental animals. This study compared fine-needle aspiration (FNA) and skin biopsy methods for obtaining bacilli and fungal cells to experimentally infect animals. Lepromas from one armadillo and one enlarged footpad of a mouse previously inoculated with L. loboi were submitted to FNA and biopsy. Materials collected were processed for inoculation in mice. Acid-fast bacilli (AFB) collected by two FNA procedures yielded 7.2×10(7) and 5.3×10(6) AFB/ml and biopsies yielded 1.58×10(8) and 3.5×10(8) AFB/ml from each leproma. Yeast-like cells of L. loboi collected by FNA yielded 1.0×10(6) fungal cells/ml and biopsy 1.0×10(7) fungal cells/ml. After 8 months, inoculated animals were sacrificed and the inoculated footpads submitted to histopathological examination and counting of AFB and fungal cells. The results obtained by the two methods were comparable for both microorganisms. Biopsy may be replaced by FNA during harvesting of material for different purposes, especially for experimental inoculation of mice in leprosy and Jorge Lobo's disease, with the advantage of FNA being a simpler, less invasive, and less costly method.
... FNA also allows the performance of histochemical, immunohistochemical, and molecular techniques for the identification of infectious agents and the searching for oncogenic proteins and specific molecular changes causing disease [9] . The results of FNA reported in the literature are comparable to the results of biopsy [10,11,12] . Out of the 34 patients of leprosy in whom histopathology was done, 27 (79.4%) ...
Article
Objectives The diagnosis of cutaneous tuberculosis is challenging due to its diverse clinical manifestations, paucibacillary state and lack of proper diagnostic tests. Clinico‐pathological correlation is still frequently used for diagnosis. There is paucity of literature on cytomorphological features. Immunochemistry can help as an ancillary test. Methods Clinical diagnosis was made after thorough history and physical examination. Modified Fine Needle Aspiration technique was used to collect cytology samples and 3 mm punch biopsy for histological examination. Findings on histopathology were compared with cytomorphology. Immunochemical staining with anti‐TB polyclonal antibody using standard Polymer‐based‐HRP immunochemistry technique and comparison of cytology and histology findings. Results The morphological spectrum of biopsy and cytology showed high correlation using nine parameters: necrosis, granulomas, giant cells, AFB, neutrophilic infiltrate, presence of lymphocytes, histiocytes, collagen bundles, and immunochemistry. Diagnostic correlation of FNA compared to biopsy was found to be 90.3%. On comparing cytomorphology of scrofuloderma and lupus vulgaris, all the parameters were found more frequently in scrofuloderma except for granulomas, giant cells and immunochemistry. Immunochemistry showed sensitivity and specificity of 90.3% and 70% on biopsy, respectively, compared to 67.7% and 60% on FNA, respectively. Combined sensitivity of IHC and ICC was 96.8%. Conclusions The cytomorphological spectrum of cutaneous tuberculosis is comparable to clinicohistopathology with a high correlation of 90.3%. However, sub classification on FNA is difficult on cytology alone. While FNAC is a better diagnostic tool for finding AFBs hence confirming the diagnosis, biopsy is better for immunochemistry. Thus, biopsy and FNA complement each other.
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Background Tuberculosis of the thyroid is very rare and does not strike the clinician as a first clinical diagnosis of a thyroid nodule. To our knowledge, only 40 cases of tuberculous thyroiditis diagnosed by fine needle aspiration cytology (FNAC) are described in the English literature. Case We report a case of tuberculosis thyroiditis in a young woman who presented with a right-side solitary thyroid nodule of short duration (15 days), diagnosed by FNAC and confirmed by positive immunocytochemistry with monoclonal antibody to Mycobacterium tuberculosis complex. Ziehl Neelsen staining for acid-fast bacilli (AFB) was negative. Conclusion FNAC provides a confident preoperative diagnosis of thyroid tuberculosis, obviating the need for unnecessary surgical removal of thyroid nodule. Immunocytochemistry is an important diagnostic adjunct to FNAC in AFB-negative cases.
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