Diagnosing adult and pediatric extrapulmonary tuberculosis by MPT64 antigen detection with immunohistochemistry and immunocytochemistry using reproduced polyclonal antibodies.

Diagnosing extrapulmonary tuberculosis (EPTB) is challenging. Immunohistochemistry or immunocytochemistry has been used to diagnose tuberculosis (TB) by detection of MPT64 antigen from various extrapulmonary specimens and has shown good diagnostic performance in our previous studies. The test can distinguish between disease caused by Mycobacterium tuberculosis (Mtb) complex and nontuberculous mycobacteria and can be applied on formalin-fixed paraffin-embedded tissue. As the antibodies previously used were in limited supply, a new batch of polyclonal antibodies was developed for scale-up and evaluated for the first time in this study. Our aim was to assess the diagnostic accuracy of the MPT64 test with reproduced antibodies in the high burden settings of Pakistan and India. Patients were enrolled prospectively. Samples from suspected sites of infection were collected and subjected to histopathologic and/or cytologic evaluation, routine TB diagnostics, GeneXpert MTB/RIF (Xpert), and the MPT64 antigen detection test. Patients were followed until the end of treatment. Based on a composite reference standard (CRS), 556 patients were categorized as TB cases and 175 as non-TB cases. The MPT64 test performed well on biopsies with a sensitivity and specificity of 94% and 75%, respectively, against a CRS. For cytology samples, the sensitivity was low (36%), whereas the specificity was 81%. Overall, the MPT64 test showed higher sensitivity (73%) than Xpert (38%) and Mtb culture (33%). The test performed equally well in adults and children. We found an additive diagnostic value of the MPT64 test in conjunction with histology and molecular tests, increasing the yield for EPTB. In conclusion, immunochemical staining with MPT64 antibodies improves the diagnosis of EPTB in high burden settings and could be a valuable addition to routine diagnostics.

Programmed Death Ligand -1 and Gene Mutation Characterization of Lung Malignancies in Patients at a Rural Hospital in Central India.

INTRODUCTION: Lung malignancy is one of the most common neoplasms worldwide. Accurate histology sub-typing and identification of gene mutations in lung tumours are considered important to administer targeted therapy for improved clinical outcome. Our aim is to determine the frequency of EGFR mutation and Programmed death ligand-1 (PD -L1) status of lung malignancies in patients attending a rural hospital in Central India. MATERIALS AND METHODS: Formalin-fixed histology diagnosed lung malignancy (n=99) bronchoscopic/trucut lung biopsies were identified and the tissue blocks and slides were retrieved. Histology typing and staging of the lesions was assessed. PD-L1 expression on biopsy was detected by immunohistochemistry using commercially available primary antibody. PD-L1 expression was assessed and semi-quantified based on the intensity and proportion of tumour cells stained for the marker. EGFR gene mutation at exon19 and 21 was detected by polymerase chain reaction of tissue from paraffin blocks. Final analysis was performed on 87 biopsies for status of EGFR mutation and PD-L1 expression. RESULTS: The average age of lung malignancies patients was 63 years, with a preponderance of males. Advance disease in stage III and stage IV was more common in squamous cell carcinoma as compared to adenocarcinoma (p < 0.01). Mutations at exon 19-21 of the EGFR gene were detected in 7/87 (8%) cases of adenocarcinoma and all of these patients were non-smokers. A total of 52.9% of biopsies showed PD-L1 expression, which was higher in adenocarcinoma patients (p=0.04), smokers (p=0.00), and stage II and III patients (p= 0.00). CONCLUSION: EGFR gene mutations at exon 19 or 21 are seen in lung adenocarcinoma cases. PD-L1 expression was observed in EGFR mutated tissues. Our results should be further validated with large sample size and multicenter clinical data before extrapolation to design immunotherapy strategies.

Significance of non-granulomatous cytomorphology on fine needle aspirate in lymphadenitis cases classified as tuberculous by using a composite reference standard.

BACKGROUND: Fine needle aspiration cytology (FNAC) is established as a first line investigation for tuberculous lymphadenitis (TBLA). We aimed to describe the various cytomorphologic features of tuberculosis (TB) on FNAC and their contribution in the diagnostic decision-making in suspected TBLA cases. METHODS: Patients with presumptive TBLA were prospectively enrolled (n = 266) and subjected to routine diagnostic work-up for TB, including FNAC samples, and followed until the end of treatment. Patients were categorized as TB or non-TB cases based on a composite reference standard of which the various cytomorphologic patterns were compared. Sensitivity, specificity, positive predictive value, negative predictive value and accuracy was calculated using cross-tabulation. RESULTS: Fifty-six patients were categorized as bacteriologically confirmed TB, 102 as clinically confirmed TB and 108 as non-TB. The most common cytomorphologic pattern among TB cases (59%) was granulomatous inflammation with necrosis, however, about one-third of tuberculous lymphadenitis patients presented with non-granulomatous inflammation, with 21% showing only necrosis and 13% presenting with a reactive pattern. The overall sensitivity and specificity of FNAC was 85% and 66%, respectively. CONCLUSIONS: We found that about one-third of TBLA patients presented without granulomas on FNA, highlighting the importance of considering TB in a wide spectrum of cytomorphology in a high TB burden setting. Our study supports the use of FNAC as a first-line investigation tool for diagnosing TBLA in a low-resource setting due to its relative simplicity and good sensitivity. However, the low specificity of FNAC, emphasizes the need for a second-tier confirmatory test with improved specificity.