Nasal swab real-time PCR is not suitable for in vivo diagnosis of bovine tuberculosis

Abstract

RESUMO.-[A PCR em tempo real de swab nasal não é adequada para o diagnóstico in vivo de tuberculose bovina.] A tuberculose bovina (bTB) é uma zoonose que causa perdas econômicas e riscos à saúde pública em muitos países. O diagnóstico da doença em animais vivos é realizado pelo teste intradérmico da tuberculina, que é baseado em reações de hipersensibilidade tardia. Como a tuberculose tem resposta imunológica complexa, este teste tem limitações em termos de sensibilidade e especificidade. Este estudo procurou desenvolver uma abordagem alternativa para o diagnóstico in vivo da tuberculose bovina, com base na reação em cadeia da polimerase (PCR) em tempo real. Bovine tuberculosis (bTB) is a zoonosis causing economic losses and public health risks in many countries. The disease diagnosis in live animals is performed by intradermal tuberculin test, which is based on delayed hypersensitivity reactions. As tuberculosis has complex immune response, this test has limitations in sensitivity and specificity. This study sought to test an alternative approach for in vivo diagnosis of bovine tuberculosis, based on real-time polymerase chain reaction (PCR). DNA samples, extracted from nasal swabs of live cows, were used for SYBR ® Green real-time PCR, which is able to differentiate between Mycobacterium tuberculosis and Mycobacterium avium complexes. Statistical analysis was performed to compare the results of tuberculin test, the in vivo gold standard bTB diagnosis method, with realtime PCR, thereby determining the specificity and sensitivity of molecular method. Cervical comparative test (CCT) was performed in 238 animals, of which 193 had suitable DNA from nasal swabs for molecular analysis, as indicated by amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, and were included in the study. In total, 25 (10.5%) of the animals were CCT reactive, of which none was positive in the molecular test. Of the 168 CCT negative animals, four were positive for M. tuberculosis complex at real time PCR from nasal swabs. The comparison of these results generated values of sensitivity and specificity of 0% and 97.6%, respectively; moreover, low coefficients of agreement and correlation (-0.029 and -0.049, respectively) between the results obtained with both tests were also observed. This study showed that real-time PCR from nasal swabs is not suitable for in vivo diagnosis of bovine tuberculosis; thus tuberculin skin test is still the best option for this purpose.

Review on Bovine Tuberculosis: An Emerging Disease Associated with Multidrug-Resistant Mycobacterium Species

“…The major limitation of mycobacterial culture is being confined to PM lesion samples, some studies suggest the use of nasal swabs as an alternative method [75]. However, Mayer et al stated that RT-PCR from nasal swabs is not suitable for in vivo diagnosis of bTB [100] Near the fibrous capsule, the inflammatory cell population consisted of macrophages, lymphocytes, and scattered neutrophils. Stage IV granulomas exhibited mostly necrosis and mineralization.…”

“…The major limitation of mycobacterial culture is being confined to PM lesion samples, some studies suggest the use of nasal swabs as an alternative method [ 75 ]. However, Mayer et al stated that RT-PCR from nasal swabs is not suitable for in vivo diagnosis of bTB [ 100 ].…”