Using genomic DNA copies to enumerate Mycobacterium tuberculosis load in macaque tissue samples

Area of Science:

• Microbiology
• Molecular Biology
• Infectious Diseases

Background:

• Accurate quantification of Mycobacterium tuberculosis (Mtb) bacterial load is crucial for tuberculosis (TB) research.
• Traditional colony forming unit (CFU) enumeration can be labor-intensive and may underestimate bacterial burden.

Purpose of the Study:

• To evaluate the efficacy of real-time quantitative PCR (qPCR) and digital PCR (dPCR) for quantifying Mtb load in non-human primate tissue samples.
• To compare PCR-based quantification with conventional CFU enumeration.

Main Methods:

• Tissue samples from Mtb-infected rhesus and cynomolgus macaques were analyzed.
• Bacterial load was quantified using CFU enumeration, qPCR, and dPCR.
• Three target genes (sigA, 16S, CFP10) were assessed for Mtb quantification.

Main Results:

• qPCR and dPCR demonstrated comparable Mtb quantification across tested gene targets.
• dPCR yielded the highest bacterial load estimates, followed by qPCR, then CFU counts.
• A significant correlation was observed between CFU counts and PCR-derived genomic copy numbers.
• PCR-based quantification was feasible primarily in samples with higher Mtb burdens.

Conclusions:

• qPCR and dPCR are effective methods for quantifying Mtb load in macaque tissues, particularly in actively infected, high-burden samples.
• PCR-based assays provide a valuable alternative to CFU enumeration in TB research.
• These molecular methods can reliably predict bacterial load in relevant preclinical models.