Tuberculosis (TB) is known to be a major health problem worldwide causing disease among millions of people every year. Major cause of tuberculosis in human is the infection with M.tuberculosiswhich usually causes pulmonary or lungs TB but an unknown number of patients are also infected with M.bovis which causes tuberculosis in humans as zoonotic agent along with its major hosts like cattle and deer. In developing countries where raw milk is used without pasteurisation there is a heavy risk of tuberculosis infection with M.bovis. TB infection with M.bovis mainly appears as extra pulmonary tuberculosis with and without specific symptoms of the disease.Diagnosis of subclinical asymptomatic tuberculosis and that of extra pulmonary tuberculosis is a difficult task and most of the time disease remains undiagnosed or misdiagnosed due to the unavailability of specific and sensitive diagnostic tool to diagnose the disease at early stage. Moreover prevalence of M.bovisinfection is not properly known.
This study was designed to measure the diagnostic value of Interferon gamma release assay (IGRA) for early and reliable diagnosis of subclinical extra pulmonary TB along with the molecular epidemiology of subclinical extra pulmonary TB to check the prevalence of M.bovisinfection. IGRA is a latest blood test with high specificity and sensitivity based on the principle of Interferon gamma released by effector T-Cell when exposed to M.tuberculosis antigens like ESAT-6 and CFP-10 in controlled in-vitro conditions. Eighty patients were selected for the study on the bases of the history of having day to day cattle contact along with feelings of sickness. Biopsy tissue samples of all the patients which were positive with IGRA were requested, however 24 out of 27 positive samples were collected and were first examined histologically. Twenty seven samples out of eighty were found positive with IGRA while 22 out of 24 samples were confirmed by histological examination as infected with MTB.
Both IGRA and histological examination are unable todifferentiate between the specie specific infection with M.tuberculosis orM.bovis for which differential amplification of specific fragments of bothof the species was done by running a multiplex PCR using M.tuberculosis specific 185 bp pncA product and M.bovis specific 500 bp segment. Genomic DNA was extracted from previously formalin fixed paraffin embedded (FFPE) tissues which requires pretreatment for deparaffinization. Xylene was used as deparaffinization agent. All of the twenty two samples positive with IGRA and histological study were found positive for M.tuberculosis infection and none of the sample was found positive for M.bovis infection. Results showeda close correlation among all three techniques with their specific benefits and limitations.
Study concluded that T.Spot TB (IGRA) is a potentially reliable test for the diagnosis of subclinical, extrapulmonary TB.Formalin Fixed Paraffin Embedded (FFPE) tissues may be used for TB diagnosis and other DNA based researches. Duplex PCR is a reliable technique for differential diagnosis of infection with different species of MTB complex, though none of the sample was found positive for M.boviswhich is may be due to small sample size of the study and it may further be studied in future researches. The research findings will help the clinicians to depend on IGRA testing for timely and reliable diagnosis of extrapulmonary subclinical tuberculosis and potential use of FFPE tissue samples as appropriate specimen for molecular based diagnosis of TB. Further studies are however, required to check the prevalence of M.bovis infection byincreasing sample size.