Immunoglobulin G response to mammalian cell entry 1A (Mce1A) protein as biomarker of active tuberculosis

Summary

Cell wall components are major determinants of virulence of Mycobacterium tuberculosis and they contribute to the induction of both humoral and cell-mediated immune response. The mammalian cell entry protein 1A (Mce1A), in the cell wall of M. tuberculosis, mediates entry of the pathogen into mammalian cells. Here, we examined serum immunoglobulin levels (IgA, IgM and total IgG) against Mce1A as a potential biomarker for diagnosis and monitoring tuberculosis (TB) treatment response. Serum samples of 39 pulmonary TB patients and 65 controls (15 healthy household contacts, 19 latently infected household contacts, 13 non-TB and 18 leprosy patients) were screened by ELISA. The median levels of all immunoglobulin classes were significantly higher in TB patients when compared with control groups. The positive test results for IgA, IgM and total IgG were 62, 54 and 82%, respectively. For comparison, routine sputum smear examination diagnosed only 26 (67%) of 39 TB cases. Sensitivities of IgA, IgM and IgG test were 59, 51.3 and 79.5%, respectively, while the specificities observed were 77.3, 83.3 and 84.4%, respectively. A significant decrease compared with baseline was also shown after TB treatment. These results suggest that circulating total IgG antibody to Mce1A could be a complementary tool to diagnosis pulmonary TB.

Introduction

Tuberculosis (TB) is a chronic bacterial infection, caused primarily by the obligate human pathogen Mycobacterium tuberculosis. Although nearly one-third of the human population is infected with M. tuberculosis, only 10% of these individuals develop active disease during their lifetime [1]. Early diagnosis and effective treatment of TB cases is the most effective tool available to control the disease.

The identification of the bacillus by microscopic examination of sputum smear or by culture has several limitations. Approximately 40% of TB patients test negative by microscopy, and culture requires a long time for the growth of M. tuberculosis, which delays diagnosis [2], [3]. Additionally, other pulmonary non-TB diseases such as cancer, pneumonia, pulmonary abscess, bronchitis and bronchiectasis may present with similar clinical symptoms and radiographic patterns [4], [5].

Therefore, a rapid diagnostic test with both high sensitivity and specificity is still needed. The most significant advance in last few years was the development of real time PCR assay (Xpert^® MTB/RIF) for detection of M. tuberculosis DNA and mutations associated with resistance to rifampicin. However, the higher cost and sophisticated infrastructure requirements have remained major barriers for their large-scale implementation for routine use. Further, the test does not eliminate the need for conventional tests, which are required to monitor treatment success and detect resistance to drugs other than rifampicin [6]. Currently the standard method to monitor treatment response is still sputum smear microscopy conversion after two months of treatment. In a meta-analysis, Horne et al. (2010) found that there was substantial heterogeneity in sputum status at 2 months, suggesting a low probability that a positive sputum specimen at any month could correctly predict the failure or relapse, making it more difficult to interpret the results [7]. Thus, this test is not very sensitive, and such a test is often not reliably performed in most TB-endemic settings [2].

A serological test may be attractive because it would be relatively rapid, reliable and cost-effective. However, several commercial serological tests provide inconsistent and imprecise results, therefore, the World Health Organization (WHO) has recommended not using any of these tests [8], [9], but encouraged further research to develop new tests with improved accuracy, especially because the serological tests might still be preferred over sputum smear microscopy. In addition, serology might also be economically attractive relative to culture and molecular tests given fewer infrastructure requirements and faster turnaround time.

M. tuberculosis survives and multiplies inside the host's macrophages by modulating the cells' antimicrobial effector response. In 1993, Arruda et al. reported that recombinant mammalian cell entry protein (Mce1A) expressed in Escherichia coli allows this non-pathogenic bacterium to invade HeLa cells and survive inside macrophages [10]. Mce1A is encoded by mce1A (Rv0169), which is one of 13 genes that comprise an operon. Shimono et al. showed that M. tuberculosis disrupted in the mce1 operon failed to elicit a strong Th1-type immune response and caused a formation of poorly organized mouse lung granulomas comprised mostly of foamy macrophages [11]. Casali et al. and Uchida et al. have showed that mce1A expression is regulated when M. tuberculosis is intracellular or in vivo [12], [13]. Taken together, these results suggest an essential role of Mce1A protein for the immunopathogenesis of TB.

In the present study, we evaluated humoral response (IgA, IgM and total IgG) of TB patient against Mce1A as a potential biomarker for diagnosing TB and monitoring TB treatment response in Salvador, Brazil, a setting with a high prevalence of TB. This is the first study conducted to develop new ELISA tests based on the Mce1A protein for serodiagnosis and monitoring treatment of human TB.