Standing of nucleic acid testing strategies in veterinary diagnosis laboratories to uncover Mycobacterium tuberculosis complex members

Abstract

Nucleic acid testing (NAT) designate any molecular approach used for the detection, identification, and characterization of pathogenic microorganisms, enabling the rapid, specific, and sensitive diagnostic of infectious diseases, such as tuberculosis. These assays have been widely used since the 90s of the last century in human clinical laboratories and, subsequently, also in veterinary diagnostics. Most NAT strategies are based in the polymerase chain reaction (PCR) and its several enhancements and variations. From the conventional PCR, real-time PCR and its combinations, isothermal DNA amplification, to the nanotechnologies, here we review how the NAT assays have been applied to decipher if and which member of the Mycobacterium tuberculosis complex is present in a clinical sample. Recent advances in DNA sequencing also brought new challenges and have made possible to generate rapidly and at a low cost, large amounts of sequence data. This revolution with the high-throughput sequencing (HTS) technologies makes whole genome sequencing (WGS) and metagenomics the trendiest NAT strategies, today. The ranking of NAT techniques in the field of clinical diagnostics is rising, and we provide a SWOT (Strengths, Weaknesses, Opportunities, and Threats) analysis with our view of the use of molecular diagnostics for detecting tuberculosis in veterinary laboratories, notwithstanding the gold standard being still the classical culture of the agent. The complementary use of both classical and molecular diagnostics approaches is recommended to speed the diagnostic, enabling a fast decision by competent authorities and rapid tackling of the disease.

The VetMAXTM M. tuberculosis Complex PCR Kit detects MTBC DNA in antemortem and postmortem samples from White rhinoceros (Ceratotherium simum), African elephants (Loxodonta africana) and African buffaloes (Syncerus caffer).

“…In those studies, major discrepancies were reported for sensitivity (63 -97%) and speci city (50 -97%) values between PCRs for tissue samples with visible and non-visible lesions (13). After the introduction of Real-Time quantitative PCR (RT-qPCR) for the detection of MTBC members, sensitivity (74 -100%) and speci city (97 -100%) values were considerably increased, respectively compared to conventional PCR (13). Observed variability between studies did not only depend on the type of PCR (conventional, nested or RT-qPCR), but also on the number of PCR targets (single-or multiple copy) and reagents, the type and number of samples included, and the DNA isolation methods.…”

“…Therefore, antemortem TB diagnosis in these species still relies on mycobacterial culture of respiratory tract samples such as bronchoalveolar lavage uid (BALF) from rhinoceros or BALF and trunk washes (TW) from African elephants. Because mycobacterial culture is regarded as an imperfect gold standard (11,12), detection of mycobacterial DNA in animal samples has been used as an alternative or ancillary diagnostic method (12,13). In humans, PCR assays have become a common molecular tool for rapid detection of TB patients, and now can provide a de nitive diagnosis (14,15).…”

“…In humans, PCR assays have become a common molecular tool for rapid detection of TB patients, and now can provide a de nitive diagnosis (14,15). During the early 1990's, many conventional PCRs had been developed and used for the direct detection of MTBC members in bovine tissue samples (13). In those studies, major discrepancies were reported for sensitivity (63 -97%) and speci city (50 -97%) values between PCRs for tissue samples with visible and non-visible lesions (13).…”

“…During the early 1990's, many conventional PCRs had been developed and used for the direct detection of MTBC members in bovine tissue samples (13). In those studies, major discrepancies were reported for sensitivity (63 -97%) and speci city (50 -97%) values between PCRs for tissue samples with visible and non-visible lesions (13). After the introduction of Real-Time quantitative PCR (RT-qPCR) for the detection of MTBC members, sensitivity (74 -100%) and speci city (97 -100%) values were considerably increased, respectively compared to conventional PCR (13).…”

“…Observed variability between studies did not only depend on the type of PCR (conventional, nested or RT-qPCR), but also on the number of PCR targets (single-or multiple copy) and reagents, the type and number of samples included, and the DNA isolation methods. To date, the largest reported study for the use of a standardised commercial RT-qPCR in cattle was the use of the VetMAX TM MTBC qPCR Kit using a Bayesian approach and reported a sensitivity of 87.7% and a speci city of 97% (13). Since then, this RT-qPCR has been used extensively in wildlife like wild boars, badgers, and deer and its use has drastically improved bTB surveillance programs in France (16)(17)(18).…”