Immunocytochemical detection of Mycobacterium Tuberculosis complex specific antigen, MPT64, improves diagnosis of tuberculous lymphadenitis and tuberculous pleuritis

Abstract

Background: A rapid, sensitive and accurate laboratory diagnosis is of prime importance in suspected extrapulmonary tuberculosis (EPTB) cases. However, traditional techniques for the detection of acid-fast bacilli have limitations. The aim of the study was to evaluate the diagnostic value of immunocytochemical staining for detection of Mycobacterium tuberculosis complex specific antigen, MPT64, in aspirates from pleural effusions and lymph nodes, the most common presentations of EPTB.

Method: A cross-sectional study was conducted by including patients at Tikur Anbessa Specialized Hospital and the United Vision Medical Services from December 2011 to June 2012. Lymph node aspirates and pleural fluid samples were collected and analyzed from a total of 51 cases (26 tuberculous (TB) pleuritis and 25 TB lymphadenitis) and 67 non-TB controls. Each specimen was subjected to Ziehl-Neelsen (ZN) staining, culture on Lowenstein- Jensen (LJ) medium, cytological examination, Polymerase Chain Reaction (PCR) using IS1081gene sequence as a primer and immunocytochemistry (ICC) with polyclonal anti-MPT64 antibody. All patients were screened for HIV.

Result: ICC was positive in 38 of 51 cases and in the 7 of 67 controls giving an overall sensitivity and specificity of 74.5% and 89.5%, respectively. Using IS1081-PCR as a reference method, the sensitivity and specificity, positive and negative predictive value of ICC was 88.1%, 89.5%, 82.2% and 93.2%, respectively. The case detection rate increased from 13.7% by ZN stain to 19.6% by LJ culture, to 66.7% by cytology and 74.5% by ICC.

Conclusion: Immunocytochemistry with anti-MPT64 antigen improved detection of TB in pleural effusion and lymph node aspirates. Further studies using monoclonal antibodies on samples from other sites of EPTB is recommended to validate this relatively simple diagnostic method for EPTB.

Figure 1

Immunocytochemical staining of lymph node aspirates (A), pleural fluid (B) and negative lymph node aspirate sample (C). Positive staining is seen as reddish brown within cells and extracellular in necrotic areas as indicated by the arrow in A and B. Pattern of the stained positive cells may be granular or diffuse.

Figure 2

IS1081-PCR of DNA extracted directly from pleural fluid and lymph node aspirate of lane 1 represents a 100 bp DNA ladder (molecular marker), Lane 2 through lane 4 are DNA extracted from pleural fluid, Lane 5 and lane 6 are DNA extracted samples from lymph node aspirates, Lane 7 thorough 9 represent an amplification product of the positive controls, i.e. M. tuberculosis strain H37Rv DNA, known ZN-positive and culture-positive test sample, and positive sample from previous PCR run which showed positive result, Lane 10 through 12, i.e. an extraction control, a reaction with substitution of Qiagen RNase free-water for the test template, and a negative sample from previous PCR assay gave a negative result.

Figure 3

Positivity of laboratory tests among cases.