Mycobacterium tuberculosis Malate Synthase- and MPT51-Based Serodiagnostic Assay as an Adjunct to Rapid Identification of Pulmonary Tuberculosis

Abstract

The 81-kDa malate synthase (MS; Rv 1837c) and the 27-kDa MPT51 (Rv 3803c) of Mycobacterium tuberculosis are immunodominant antigens recognized by serum antibodies from ϳ80% of human immunodeficiency virus-negative smear-positive tuberculosis patients from India. We now provide evidence that the use of the MS/MPT51-based serodiagnostic assay can serve as an adjunct to sputum microscopy in the rapid diagnosis of pulmonary tuberculosis.Approximately 50% of the pulmonary tuberculosis (PTB) cases in the United States are smear negative for acid-fast bacilli (AFB), leading to a diagnostic delay of several weeks due to the low growth rate of Mycobacterium tuberculosis (2a). Delayed recognition of smear-negative PTB is estimated to be responsible for ϳ20% of the cases of TB transmission (2). Nucleic acid amplification assays can detect ϳ50% of these cases but are too expensive for routine screening for smearnegative PTB (1,13,20). Diagnostic tests that can enhance the early recognition of paucibacillary TB are therefore urgently required.Our earlier studies have delineated a subset of ϳ10 to 12 culture filtrate proteins of M. tuberculosis that are recognized by antibodies (Abs) in patients with human immunodeficiency virus-negative (HIV Ϫ ) multibacillary as well as paucibacillary TB and in HIV-infected (HIV ϩ ) TB patients (19). Two of these, the 81-kDa protein malate synthase (MS; Rv 1837c) and the 27-kDa protein MPT51 (Rv3803c) are recognized by Abs in ϳ80% of HIV Ϫ , smear-positive TB patients from India (21). Our studies have shown that paucibacillary TB patients have low titers of Abs compared to multibacillary patients (19). The goal of the present study was to determine the presence of Abs to MS and/or MPT51 in culture-confirmed patients from the United States, who tend to present at an early stage of TB, and to assess the ability of the serodiagnostic assay to serve as an adjunct to AFB smears for the rapid diagnosis of PTB.Approval for human subject research was obtained from the institutional review boards of the New York University School of Medicine, Bellevue Hospital, and the Manhattan Veterans Administration, and written informed consent was obtained from all individuals prior to the drawing of blood samples and review of medical records. Patients clinically suspected of having PTB were enrolled, and their sera were frozen. Only specimens from patients later confirmed to be M. tuberculosis culture positive (TB ϩ ; n ϭ 53) were included in the studies (Table 1). Subjects treated for TB for Ͼ14 days were excluded. Serum specimens from 55 healthy controls with no risk factors for HIV infection (36 with tuberculin skin test [TST]-positive results and 19 with TST-negative results) were also obtained. The TST-positive subjects had indurations of Ͼ10 mm and included volunteers who had been vaccinated with Mycobacterium bovis BCG and/or had a history of TB exposure. Sera from 40 asymptomatic HIV ϩ patients, all of whom were on highly active antiretroviral therapy, were also included; 36/40 (90%) had undetectable...

Antibodies against Immunodominant Antigens of Mycobacterium tuberculosis in Subjects with Suspected Tuberculosis in the United States Compared by HIV Status

“…The Mann-Whitney U test was used for two-group comparisons of median antibody reactivities. * , P Ͻ 0.05; ** , P Ͻ 0.01; *** , P Ͻ 0.001. ϩ /TB patients in the current cross-sectional study is similar to the combined sensitivity with both antigens in our earlier case-control study, 77 and 80%, respectively (1). The confirmation of the high sensitivity in the U.S., where TB can be diagnosed early due to the use of multiple technologies, and the high Ab responses in Indian HIV ϩ /TB patients together emphasize the value of these antigens in the rapid diagnosis of TB in countries where TB is rampant in HIV ϩ patients.…”

“…Moreover, there was no difference of Ab responses to either antigen between asymptomatic U.S. controls and TST Ϫ and TST ϩ individuals from settings in which TB is endemic. As was observed in the prior U.S. case-control study, both HIV Ϫ /TB and HIV ϩ /TB patients had significantly higher Ab reactivities than subjects with LTBI, and U.S. HIV ϩ /TB demonstrated significantly higher reactivities irrespective of CD4 ϩ T-cell counts, viral loads, or sputum smear results compared to those of HIV Ϫ /TB patients (1,26,37). The evaluation of Ab reactivities in TB suspects with ORD revealed higher values in U.S. HIV ϩ /TB than U.S. HIV ϩ /ORD patients, while no such differences were observed among U.S. HIV Ϫ TB suspects.…”

“…Similar studies with U.S. patients demonstrated that while anti-MS and/or anti-MPT51 Abs were present in only ϳ40% of the HIV Ϫ patients at early stages of TB, ϳ80% of the U.S. HIV ϩ TB patients were Ab positive (1). Nevertheless, combining serology with sputum microscopy improved the detection of TB in both groups compared to that of microscopy alone, and it led to the identification of 90% of HIV ϩ TB patients, compared to 60% by microscopy alone (1). These findings are of high clinical relevance, since the rapid identification and treatment of early TB is crucial for HIV ϩ patients, in whom the dual infection leads to the acceleration of both diseases (20,35).…”

“…In contrast, positive antibody (Ab) responses to several new antigens during active TB but not LTBI have been reported. However, most studies have focused on patients with advanced TB, and the utility of these responses appears to be limited in patients with low mycobacterial burden (reviewed in references 29 and 30).Two immunodominant M. tuberculosis proteins, the 81-kDa malate synthase (MS; Rv 1837c, GlcB) and the 27-kDa MPT51 (Rv3803c), are reported to elicit Ab responses during early and advanced stages of TB in both HIV Ϫ and HIV ϩ patients (1,2,10,16,24,27,37). This is important because HIV ϩ TB patients appear to develop Ab responses to a smaller repertoire of M. tuberculosis antigens than that of HIV Ϫ TB patients (24,25).…”

Correlation between Serum and Plasma Antibody Titers to Mycobacterial Antigens

“…Much progress has been made in the field of TB serology in the past decade (10,13,15; reviewed in reference 16), and recent studies have indicated that serodiagnosis in the form of detection of antibodies (Abs) to immunodominant antigens of Mycobacterium tuberculosis could be a useful adjunct test to diagnose TB earlier (2,3,17). Traditionally, serum is used to test for Abs to mycobacterial antigens.…”