Sudan Journal of Medical Sciences Volume 17, Issue no. 3, DOI 10.18502/sjms.v17i3.12103 Production and Hosting by Knowledge E Research Article Evaluation of Fine-needle Aspiration Cytology (FNAC) Sensitivity Compared to PCR for Diagnosing Tuberculosis Lymphadenitis Omar Mustafa1, Ebtehal Mohamed2, Ahmed Omer2, Abdelmonem Mohamed2, Sheima Abdemagid3, Alaa Ali4, Nafisa Hassan5, Mayada Khalil6, and Nagia Suliman6 1Department of Histopathology and Cytology, College of Medical Laboratories Science, University of Medical Science and Technology (UMST) 2Department of Clinical Chemistry, College of Medical Laboratory Science, Al-Neelain University 3Department of Microbiology, College of Medical laboratory science, University of Alzaiem Al Azhari 4Department of Hematology and Immunohematology, College of Medical Laboratory Science, Sudan University of Science and Technology 5Department of Clinical Chemistry, College of Medical Laboratory Science, University of El imam El Mahdi 6Department of Medical Microbiology, College of Medical Laboratory Science, University of Gezira ORCID: Abdelmonem Mohammed: https://orcid.org/0000-0002-3925-5268 Ahmed Omer: https://orcid.org/0000-0001-9035-5558 Mayada Khalil: https://orcid.org/0000-0001-7619-097X Shiema Abdelmagid: https://orcid.org/0000-0003-0090-1514 Alaa Ali: https://orcid.org/0000-0002-7482-7065 Nagia Suliman: https://orcid.org/0000-0002-0072-9315 Ebtehal Mohamed: https://orcid.org/0000-0003-2690-1248 Nafisa Hassan: https://orcid.org/0000-0002-2923-0198 Omer Mustafa: https://orcid.org/0000-0002-2540-1877 Abstract Background: Tuberculosis (TB) is a major healthcare burden in Sudan and other developing countries, it is considered the second most common cause of death from infectious diseases after those due to AIDS. In Sudan, TB lymphadenitis (TBLA) remains one of the major health problems. This descriptive cross-sectional study was conducted at the University of Medical Sciences and Technology (UMST) and Total Labcare Diagnostic Center (TDC). The study aims to compare the sensitivity of Fine Needle Aspiration Cytology (FNAC) smears with that of the Polymerase Chain Reaction (PCR) for the diagnosis of TBLA. Methods: Fifty-five dry smears were obtained using fine-needle aspiration (FNA) from an enlarged lymph node. PCR was applied to detect the target gene (IS6110). May- Grunwald-Giemsa (MGG) or Diff quick stains were used. Results: Two (4%) patients with TBLA were non-necrotic, while fifty-three of them (96%) were necrotic. Moreover, 17 (30%) fine-needle lymph node aspiration specimens were confirmed by PCR to be positive for Mycobacterium tuberculosis complex (MTB complex) while 38 (70%) of them were negative. Conclusion: There was no significant difference between the sensitivity of PCR and that of FNAC (P-value = 0.33). How to cite this article: Omar Mustafa, Ebtehal Mohamed, Ahmed Omer, Abdelmonem Mohamed, Sheima Abdemagid, Alaa Ali, Nafisa Hassan, Mayada Khalil, and Nagia Suliman (2022) “Evaluation of Fine-needle Aspiration Cytology (FNAC) Sensitivity Compared to PCR for Diagnosing Tuberculosis Lymphadenitis,” Sudan Journal of Medical Sciences, vol. 17, Issue no. 3, pages 330–340. DOI 10.18502/sjms.v17i3.12103 Page 330 Corresponding Author: Nagia Suliman; email: nagisuliman@hotmail.com Received 20 December 2021 Accepted 21 May 2022 Published 30 September 2022 Production and Hosting by Knowledge E Omar Mustafa et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. Editor-in-Chief: Prof. Nazik Elmalaika Obaid Seid Ahmed Husain, MD, M.Sc, MHPE, PhD http://www.knowledgee.com https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ Sudan Journal of Medical Sciences Omar Mustafa et al Keywords: tuberculosis, lymphadenitis, lymph node, FNAC, PCR 1. Introduction Tuberculosis (TB) is a common infectious agent associated with prominent levels of morbidity and mortality, especially in developing countries [1, 2]. Globally, in 2018, about 10 million TB cases and 1.5 million TB deaths were estimated [3]. In Africa, the prevalence of TB remains as one of the major health problems due to malnutrition, poverty, and poor diagnosis. Based on a previously published report, about 30–40% of HIV patients die from TB in African countries [4]. Sudan is considered one of the endemic areas of TB. According to the WHO, in 2013, 20,181 TB cases were detected, of which 980 (30%) were new sputum smear-positive cases. However, there are many unreported cases due to the low-quality system of data reporting [5, 6]. One of the WHO TB strategies after 2015 is global reduction in TB epidemic death and incidence rate of up to 90% and 95%, respectively [7]. TB is caused by a bacterium called Mycobacterium tuberculosis, which is a member of M. tuberculosis complex (M. tuberculosis, M. bovis, M. microti, and M. africanum). There are two forms of clinical TB – pulmonary TB (PTB) which usually attacks the lungs and extrapulmonary TB (EPTB) that attacks other organs such as the kidneys, spine, and brain [8]. However, the major EPTB is TBLA. It causes an enlargement of lymph nodes caused by infection or inflammation [9]. Both the diagnosis and therapy for TBLA represent a challenge because it has physical and laboratory findings feature similar to other pathologic processes [10]. It is difficult to diagnose TBLA by routine methods such as the microscopic Ziehl-Neelsen (ZN) stain and microbiology culture in the Lowenstein-Jensen medium. Among the most practical applications for cytological analysis of lymph node aspirates is fine needle aspiration cytology (FNAC) [11, 12]. Fine needle aspiration (FNA) is accepted by most patients as a noninvasive method and is considered by pathologists for evaluating lymphadenopathy and preserving lymph node structure [13]. Enlarged lymph nodes are a prime target for FNA. Although, as mentioned in the previously published studies, cytology can provide a definitive morphological prognosis of lymphadenopathy, but combination with con- firmatory techniques is recommended. In the current study, the molecular technique used is polymerase chain reaction (PCR) to detect the M. tuberculosis complex. PCR is the molecular tool that permits the exponential amplification of target DNA [14]. The use of PCR to diagnose mycobacterial infection is not a novel procedure, however, PCR still represents a gold standard for molecular techniques and adds diagnostic value DOI 10.18502/sjms.v17i3.12103 Page 331 Sudan Journal of Medical Sciences Omar Mustafa et al for suspicious results. The goal of this study was to assess the sensitivity of FNAC technique compared to PCR in confirming TBLA. 2. Materials and Methods A descriptive cross-sectional laboratory-based research was carried out at the University of Medical Sciences and Technology and Total Labcare Diagnostic Center. Fifty-five patients were included from both genders of different age groups. Fine needle lymph node aspiration specimens were collected under all aseptic precautions, using standard disposable 27-gauge needles (Figure 1). High-quality smears were prepared and stained with Diff-Quick (Romanowski stain). In the staining procedure of Figure 1: Collection of a sample trapped in the hub of the biopsy needle, the sample was aspirated with another needle mounted on a syringe. Diff-Quick, air-dried smears are fixed in Diff-Quick fixative (or methanol) for 30 sec followed by stain with Diff-Quick solution I for 30 sec, and then stain with Diff-Quick solution II for 30 sec. Then, they are rinsed in tap water to remove excess stain and rapidly dehydrated in absolute alcohol. After that the slides are cleared and mounted. 2.1. DNA extraction DNA was extracted by scraping lymph node smears material. Cinnagen, Iran Kit was used, the kit contains lysis buffer to rupture and release the cells’ constituents, the pre- cipitation solution was used to precipitate proteins with other substances, but the DNA flooded in the mixture. This method used column tube which contains silica particles plate, positive charge of the silica attached to DNA and passed other substances. The DOI 10.18502/sjms.v17i3.12103 Page 332 Sudan Journal of Medical Sciences Omar Mustafa et al elution buffer of this kit was warmed at 64ºC, which eluded the DNA and the DNA was obtained at the bottom of new 1.5 ml eppendorf tubes. 2.2. PCR reagents DNA template; PCR master mix contains TaqTMDNA Polymerase (5U/μl), dNTPs 2.5 mM each, reaction buffer (10×); stock primers (forward and reverse primers were 5′-GCCTACGTGGCCTTTGTCAC-3′ and 5′-C3-GTCCAGATGGCTTGCTCGAT-3′ respec- tively [15]; DNA MTB-positive; DDH2O (Double Distilled water); and gel loading dye (1×). 2.3. PCR protocol Extracted DNA was brought at –20ºC, thawed, and kept on ice cryo-rack for processing. At the same time, stock primers, dNTPs, and reaction buffer were brought at room temperature (RT) and kept on ice cryo-rack for thawing. Sterile PCR water was brought out from refrigerator and aliquoted on 1.5-ml tubes. 2.3.1. PCR optimization Small fraction of positive control (brought from tuberculosis unit in the national health laboratory) was first subjected to PCR amplification. After successful amplification, the rest of the samples were analyzed. In the events of the negative results, samples were diluted from the outer product up to one in hundred and the original DNA was diluted up to tenfold to minimize the inhibitors. 2.3.2. PCR procedure In PCR room at the Biological Safety Cabinet Class II, the maxim PCR premix tubes (20 µl) and the DNA sample both stored at –20ºC were taken out and, respectively, placed on ice and bench for thawing. Next, 2-µl TB primer mix (forward and reverse) was added to each maxim tube and 13-µl ddH2O was added to the sample tube and positive control while 18-µl ddH2O was added to the negative control. Moreover, 5-μl DNA was added to each tube (filter tips were used), vortex for 5 sec and all samples were run in PCR machine. DOI 10.18502/sjms.v17i3.12103 Page 333 Sudan Journal of Medical Sciences Omar Mustafa et al 2.3.3. PCR amplification of MTB gene The amplification was carried out using 10× PCR buffer (10 mMtris-Hcl, Ph 8.3, 50 mM KCL, 1.5 mMMgcl2); 2.5mM of each dNTPs, DNA template and i-TaqTMDNA Polymerase (5U/μl) in a final volume 20 μL. Reactions were performed in a thermal cycler TC-412 with the following thermal profile: primary denaturing at 94ºC for 5 min, denaturing at 94ºC for 30 sec, annealing temperature at 62ºC for 30 sec, extension at 72ºC for 1 min, and a final extension at 72ºC for 30 sec for 40 cycles for the outer PCR. Then the PCR products were examined in the agarose gel. 2.4. Electrophoresis protocol 2.4.1. 10X TBE Tris-Borate-EDTA (TBE) buffer preparation The working solution of 1X TBE was prepared from the stock solution (1 L) which contained the following: 89 mMTris base, 89 mM boric acid, and 2 mM EDTA. It was used for agarose gel preparation and as a running buffer for electrophoresis. 2.4.2. Preparation of DNA loading dye solution, (bromophenol blue) The dye was prepared as follows: 0.25 gm of bromophenol blue (SIGMA), 50% pure glycerol (10 ml) and 0.4 m EDTA. It was then mixed and stored in a brown bottle at 4ºC. 2.4.3. Preparation of 100 base pairs ladder DNA ladder was prepared for electrophoresis as follows: 5 μl DNA ladder (SIGMA), 5 μl water, and 2 μl gel loading dye, the mixture was stored at –20ºC. 2.4.4. Preparation of agarose gel for PCR product (2%) In clean dry bottle, 50 ml of 1X TBE buffer was added, 1 gram of agarose powder was then added to the bottle. The powder was dissolved by heating the solution in a microwave, and then cooled at room temperature (until 50ºC). Next, the solution was poured in a gel tray with two combs row (well-maker) until completely solidified. In the running tank, 500 ml of 1X TBE buffer was added with 1 µl of DNA-safe stain. Agarose gel plate was placed in the running direction and the first well was loaded with DNA marker (100 pb) and the second with negative control. Samples were loaded in the next DOI 10.18502/sjms.v17i3.12103 Page 334 Sudan Journal of Medical Sciences Omar Mustafa et al wells, and the last well was loaded with positive control. The power supply was adjusted to 100mA for 30 min. Finally, the gel plate was transferred to the gel documentation system to visualize the DNA and photograph the bands. 3. Results The study involved 55 patients, 24 male and 31 female, with an average age of 37.5 years. All cases showed enlarged cervical lymph nodes. Fine needle aspiration for cytological examination showed that 53 (96%) cases had TBLA with necrosis and granulomatous inflammation with or without detection of epithelioid cells, while 2 cases showed non-necrotizing granulomatous lymphadenitis with only epithelioid and inflammatory cells detected (Figures 1 & 2). Products of PCR on gel electrophoresis are shown in Figure 4. The correlation between cytological findings and PCR results are set out in Table 1, showing a Chi-square with p-value > 0.05 which was considered statistically insignificant (<0.05 was considered significant). Figure 2: Tuberculosis lymphadenitis with necrotizing shows granulomas variable in size with a mixture of epithelioid macrophages and lymphocytes. ‘ Chi-square p-value was obtained = 0.335∗. DOI 10.18502/sjms.v17i3.12103 Page 335 Sudan Journal of Medical Sciences Omar Mustafa et al Figure 3: Tuberculosis lymphadenitis with necrotixing material and a few degenerating nuclei (Romanowsky stain). Figure 4: PCR products on gel electrophoresis under UV light. Upper row right of gel (M): DNA marker; L1: positive control; L7: negative control; L4–L6: positive samples for MTB complex; L2 and L3: negative for MTB complex. 4. Discussion PCR is considered as one of the confirmatory methods for TB. There are other molecular techniques such as Nucleic Acid Amplification Test (NAAT) and GeneXpert (GXP) both DOI 10.18502/sjms.v17i3.12103 Page 336 Sudan Journal of Medical Sciences Omar Mustafa et al Table 1: Comparison of overall sensitivity of cytology and PCR. Cross-tabulation PCR Total Positive Negative Cytology Necrotizing 17 36 53 (30%) (66%) (96%) Non-necrotizing 0 2 2 0 (4%) (4%) Total 17 38 55 (30%) (70%) (100%) of which depend on the principle of nucleic acid detection of TB. But in the current setting, the PCR technique was used as a confirmatory method for FNAC results. In the present study, 55 patients were enrolled with a suspicion of cervical TBLA. Fine needle lymph node aspirate was collected and examined cytologically. Fifty-three smears showed necrotic material while two were non-necrotic. All these smears were diagnosed as cervical TBLA. PCR was done and 17 (30%) specimens were positive and 38 (70%) were negative for MTB complex. Our specimens have shown lower PCR- positivity numbers which may be attributed to many factors, including the small amount of specimen and subsequently fewer organisms (especially, after splitting the specimen for cytological assays), internal variations in the DNA extraction and concentration. The storage temperature of the specimen could also affect the sensitivity, especially in tropical areas where the temperatures are high [16]. While our cytological findings have shown higher positivity of 53 (96%) smears with necrosis this may be due to their larger size and well-stained smears on the slide. Statistically, there was an insignificant difference between the PCR sensitivity compared to the FNAC with a P-value of 0.33. PCR-positive specimens also were positive for cytological smears diagnosed as TBLA. Thus, our results agree with Chantranuwat et al. (2006), Tansuphasiri et al. (2004) [17, 18]. 5. Conclusion There was no significant difference between the FNAC and PCR sensitivities in the diagnosis of TBLA. All PCR-positive cases also showed cytological positivity for TBLA. PCR could be used as a confirmatory test for MGG or Diff-Quick-stained cytological smears. PCR could be used as a practical and valuable method when no smear spec- imen is available. However, a molecular technique like PCR is costly and unavailable in a limited resource setting. So, FNAC could be an effective diagnostic technique for DOI 10.18502/sjms.v17i3.12103 Page 337 Sudan Journal of Medical Sciences Omar Mustafa et al TBLA, because it is easy to perform, cost-effective, and convenient, especially in high prevalence areas in poor economies with limited resources. Acknowledgements The authors are thankful to Dr. Alfatih Aboalbasher Yousif and Dr. Maha Mahgoub for their logistic support. Ethical Considerations Informed consent was obtained from all patients. Competing Interests None declared. 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DOI 10.18502/sjms.v17i3.12103 Page 340 Introduction Materials and Methods DNA extraction PCR reagents PCR protocol PCR optimization PCR procedure PCR amplification of MTB gene Electrophoresis protocol 10X TBE Tris-Borate-EDTA (TBE) buffer preparation Preparation of DNA loading dye solution, (bromophenol blue) Preparation of 100 base pairs ladder Preparation of agarose gel for PCR product (2%) Results Discussion Conclusion Acknowledgements Ethical Considerations Competing Interests Availability of Data and Material Funding References