Environmental Risk Factors of Tuberculosis and Detection of Infection and Drug Resistance Through Multiplex Analysis

Abstract

Over 9 million new active tuberculosis (TB) cases emerge each year from an enormous pool of 2 billion individuals latently infected with Mycobacterium tuberculosis (M. tb.) worldwide. About 3 million new TB cases per year go undiagnosed, and 1.5 million die (equivalent to AIDS related deaths). Involvement of various host related and environmental risk factors increasing the susceptibility of the disease and emergence of MDRTB has made the control and eradication of the disease difficult. TB, however, is generally curable if diagnosed correctly and in a timely manner. The current diagnostic methods for TB and MDRTB, including state-of-the-art molecular tests, have failed in delivering the capacity needed to curtail this ongoing pandemic. Highly efficient, accurate, cost effective and scalable diagnostic tests are critically needed. We report a multiplex TB serodiagnostic panel for TB infection and a DNA based tests for MDRTB, based on microbead suspension array. Our serodiagnostic panel demonstrates sensitivity of 91% for confirmed TB cases on serum/plasma samples from patients. Sensitivities of this new test format for sputum smear positive and negative cases are 95%, and 89%, respectively, and this test has specificity of 96%. Similarly our DNA based test for MDRTB mutation has 86% sensitivity for Rifampicin, 92% for Isoniazid, 62% for Streptomycin and 63% for Ethambutol resistant samples. This test has shown 100% specificity and 100% correlation between sputum and culture samples of MDRTB. These diagnostic criteria for the multiplex TB & MDRTB test are greatly superior to that of the frontline sputum smear test (30-70% sensitivity) and conventional culture and DST. The test can be run on either blood plasma/serum or dried blood spots (DBS) for TB infection and on DNA isolated from sputum/culture of MDRTB sample, enabling use in almost any setting with scalability from 1 to 360 patients per day, and is amenable to automation for higher (1000s per day) throughput. We also have reported the involvement of well-established host related, environmental and social risk factors in susceptibility of the disease. For host-related factors, TB was found to be significantly associated with male gender, marital status, smoking, drinking, personal and family history of TB, asthma and diabetes (OR: 1.08, 1.96, 1.21, 4.26, 2.07, 3.16, 3.43 and 3.67) respectively with P-value <0.001. For environmental and socio economic factors TB was found to be associated with adult crowding, increased family size, poor ventilation and use of biofuels (OR: 4.60, 1.75, 3.29 and 3.90) illiteracy, unawareness of the disease, migration and presence of animals in the house (OR: 1.74, 0.07, 1.83 and 1.60) respectively with p-values of <0.005. Thus in combination with information of demographic details, risk factors information and use of the proposed test, diagnosis of TB and MDRTB can be possible in one day without any delay in initiation of the treatment. This approach can help TB control program to control the disease by diagnosing it efficiently, accurately, and timely