Rapid Diagnosis of Early TB in HIV+ Patients

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DESCRIPTION (provided by applicant): TB diagnosis in India relies on clinical symptoms, microscopic examination of smears made directly from sputum and/or chest X-rays (CXR). The immune dysfunction caused by HIV-infection alters clinical and radiological presentation of TB, and microbiological confirmation of TB is problematic since HIV+TB+ patients have increased frequency of smear-negative (SN) TB and extrapulmonary TB (EPTB). Despite being totally treatable with the existing drugs, the poorer performance of insensitive diagnostic tests in HIV+ patients leads to significant under-diagnosis of TB in the very population where TB progression is rapid and often fatal. A simple, rapid test to identify smear-positive (SP) and SN symptomatic or asymptomatic TB in HIV+ patients would have significant impact on TB-related morbidity and mortality. Five immunogenic proteins of M. tuberculosis that are potential candidates for devising a simple diagnostic test for TB in HIV+ patients have been identified. These are MS, MPT51, PPE55, ESAT6 and CFP10. Antibodies (Abs) to these antigens have been demonstrated at all levels of immune-suppression, regardless of pulmonary manifestations and site of TB in HIV+TB+ patients, and are also detectable in sera of asymptomatic HIV+ patients who progressed to symptomatic TB in the subsequent 2-6 months. Absence of Abs to these antigens in retrospective sera from CD4 T cell-matched HIV+ patients who did not eventually develop TB suggests that in HIV+ patients, these Ab responses signal active TB whether or not clinical symptoms of TB are present and before the bacterial burden reaches the threshold of detection by direct microscopy. In the current studies we propose to evaluate the potential of these Abs to identify SN and SP symptomatic/asymptomatic HIV+TB+ patients in prospective studies and to map their dominant epitopes that are recognized by Abs in Indian patients. For this, we will recruit a cohort of ~200 ART-na¿ve HIV+ patients and investigate them for TB using direct microscopy as well as with decontaminated/concentrated sputum smears, cultures on solid media and NAAT evaluation of sputum, blood and urine (Aim 1); evaluate the presence of Abs to recombinant purified MS, MPT51, ESAT6, CFP10 and PPE55 in serum specimens from the recruited individuals and correlate the presence of Abs with bacteriological confirmation of TB (Aim 2) and identify the immunodominant epitopes on the 5 antigens recognized by serum Abs from Indian patients on peptide microarrays to define peptides that can be used to devise a peptide-based rapid test for TB in Indian HIV+ patients. PUBLIC HEALTH RELEVANCE: The immune dysfunction caused by HIV-infection alters clinical and radiological presentation of TB, and microbiological confirmation of TB is problematic since HIV+TB+ patients have increased frequency of smear- negative (SN) TB and extrapulmonary TB (EPTB). Despite being totally treatable with the existing drugs, the lack of appropriate diagnostic tools results in identification of less than half of the HIV+TB+ patients being diagnosed before death in TB-endemic countries. More sensitive diagnostic tests like microscopy with decontaminated and concentrated smears, culture of M. tb and PCR-based nucleic acid-amplification tests (NAAT) significantly enhance the identification of SN patients. However, routine implementation of these advanced diagnostic techniques is not economically feasible in high-burden countries. Studies over the last 15 years have led to the identification, cloning, and evaluation of a panel of well-defined highly immunogenic proteins of M. tb that are potential candidates for devising a simple diagnostic test for TB in HIV+ patients whether or not clinical symptoms of TB are present and before the bacterial burden reaches the threshold of detection by direct microscopy. The goal of the current studies is to evaluate the ability of Abs to these antigens to detect SN and SP symptomatic/asymptomatic HIV+TB+ patients in prospective studies. We will also map the dominant epitopes of these proteins that are recognized by Abs in Indian patients that may be used to devise a peptide-based rapid diagnostic test for HIV+TB+ patients.