Both brucellosis and tuberculosis are chronic-debilitating systemic granulomatous diseases with a high incidence in many countries in Africa, Central and South America, the Middle East and the Indian subcontinent. Certain focal complications of brucellosis and extrapulmonary tuberculosis are very difficult to differentiate clinically, biologically and radiologically. As the conventional microbiological methods for the diagnosis of the two diseases have many limitations, as well as being time-consuming, multiplex real time PCR (M RT-PCR) could be a promising and practical approach to hasten the differential diagnosis and improve prognosis.
We designed a SYBR Green single-tube multiplex real-time PCR protocol targeting bcsp31 and the IS711 sequence detecting all pathogenic species and biovars of Brucella genus, the IS6110 sequence detecting Mycobacterium genus, and the intergenic region senX3-regX3 specifically detecting Mycobacterium tuberculosis complex. The diagnostic yield of the M RT-PCR with the three pairs of resultant amplicons was then analyzed in 91 clinical samples corresponding to 30 patients with focal complications of brucellosis, 24 patients with extrapulmonary tuberculosis, and 36 patients (Control Group) with different infectious, autoimmune or neoplastic diseases. Thirty-five patients had vertebral osteomyelitis, 21 subacute or chronic meningitis or meningoencephalitis, 13 liver or splenic abscess, eight orchiepididymitis, seven subacute or chronic arthritis, and the remaining seven samples were from different locations. Of the three pairs of amplicons (senX3-regX3+ bcsp3, senX3-regX3+ IS711 and IS6110+ IS711) only senX3-regX3+ IS711 was 100% specific for both the Brucella genus and M. tuberculosis complex. For all the clinical samples studied, the overall sensitivity, specificity, and positive and negative predictive values of the M RT-PCR assay were 89.1%, 100%, 85.7% and 100%, respectively, with an accuracy of 93.4%, (95% CI, 88.3—96.5%).
In this study, a M RT-PCR strategy with species-specific primers based on senX3-regX3+IS711 sequences proved to be a sensitive and specific test, useful for the highly efficient detection of M. tuberculosis and Brucella spp in very different clinical samples. It thus represents an advance in the differential diagnosis between some forms of extrapulmonary tuberculosis and focal complications of brucellosis.
Both brucellosis and tuberculosis are systemic infections which may involve any organ. When they affect specific locations, extrapulmonary tuberculosis and brucellosis cause symptoms that are very difficult to differentiate clinically. Mycobacterium tuberculosis complex and Brucella spp are slow-growing microorganisms whose culture and isolation require several days to weeks. Methods based on the polymerase chain reaction (PCR) have proven more sensitive than conventional culture, for both extrapulmonary tuberculosis and focal complications of brucellosis. Multiplex real time PCR is a variant of PCR in which two or more target sequences can be simultaneously amplified in a single tube. We developed and evaluated the results of several multiplex real-time PCR strategies for the rapid differential diagnosis between extrapulmonary tuberculosis and focal complications of brucellosis. Multiplex real-time PCR targeting of SenX3-RegX3+IS711 sequences showed a sensitivity of 89.1% and a specificity of 100% when applied to 91 clinical specimens. These findings provide solid evidence suggesting that multiplex real-time PCR could be a useful tool to reduce the time required for the differential diagnosis between extrapulmonary tuberculosis and complicated brucellosis, thereby improving prognosis.