Sensitivity of different DNA extraction methods and PCR to detect resistance in patients with leprosy stratified by the bacilloscopic index.

INTRODUCTION: Antimicrobial resistance in leprosy is an emerging problem, and the quantitative impact of low bacilloscopic indexes (BIs) on the sensitivity of molecular tests is unknown. We aimed to evaluate the sensitivity of gene sequencing for the detection of mutations related to antimicrobial resistance in Mycobacterium leprae in patients with low BIs using an analytical model. METHODS: Patients with leprosy were included and divided into two groups depending on their BIs (≥ 2+ and < 2+). The sensitivities of the two DNA extraction methods were compared after amplifying and sequencing the repetitive element (RLEP), folP1, rpoB and gyrA in M. leprae. RESULTS: We included 56 patients with leprosy: 35 had BIs less than 2+ (22 had negative slit-skin smear [SSS] results) and 21 patients had BIs greater than or equal to 2+. The sensitivity of the amplification of the RLEP target and the gene sequencing of folP1, rpoB and gyrA was 50 to 70% lower in patients with a BI less than 2+ and was significantly reduced in patients with lower BIs for all targets (p < 0.001). One patient had a mutation in the folP1 gene, and 14 patients had mutations in the gyrA gene, but no mutations related to antimicrobial resistance were found. CONCLUSIONS: We can conclude that the sensitivity of molecular tests is directly related to the BI, but these tests can still detect up to 20% of the targets in patients with BIs < 2+. New strategies to improve the sensitivity for detecting antimicrobial resistance in leprosy patients and reasonable clinical criteria for follow-up and the introduction of alternative treatments must be developed.

Sensitivity of different DNA extraction methods and PCR to detect resistance in patients with leprosy stratified by the bacilloscopic index

ABSTRACT Introduction: Antimicrobial resistance in leprosy is an emerging problem, and the quantitative impact of low bacilloscopic indexes (BIs) on the sensitivity of molecular tests is unknown. We aimed to evaluate the sensitivity of gene sequencing for the detection of mutations related to antimicrobial resistance in Mycobacterium leprae in patients with low BIs using an analytical model. Methods: Patients with leprosy were included and divided into two groups depending on their BIs (≥ 2+ and < 2+). The sensitivities of the two DNA extraction methods were compared after amplifying and sequencing the repetitive element (RLEP), folP1, rpoB and gyrA in M. leprae. Results: We included 56 patients with leprosy: 35 had BIs less than 2+ (22 had negative slit-skin smear [SSS] results) and 21 patients had BIs greater than or equal to 2+. The sensitivity of the amplification of the RLEP target and the gene sequencing of folP1, rpoB and gyrA was 50 to 70% lower in patients with a BI less than 2+ and was significantly reduced in patients with lower BIs for all targets (p < 0.001). One patient had a mutation in the folP1 gene, and 14 patients had mutations in the gyrA gene, but no mutations related to antimicrobial resistance were found. Conclusions: We can conclude that the sensitivity of molecular tests is directly related to the BI, but these tests can still detect up to 20% of the targets in patients with BIs < 2+. New strategies to improve the sensitivity for detecting antimicrobial resistance in leprosy patients and reasonable clinical criteria for follow-up and the introduction of alternative treatments must be developed.

Standardization of SYBR Green-Based Real-Time PCR Through the Evaluation of Different Thresholds for Different Skin Layers: An Accuracy Study and Track of the Transmission Potential of Multibacillary and Paucibacillary Leprosy Patients.

The development of new molecular techniques is essential for the early diagnosis of leprosy. Studies in the field have failed to elucidate the performance of these tests in clinical practice. We aimed to design a new primer pair for the repetitive element (RLEP) target of Mycobacterium leprae and to test the accuracy of SYBR green-based real-time PCR through the evaluation of different thresholds for different skin layers. We also aimed to track the transmission potential of multibacillary and paucibacillary leprosy patients. The in vitro validation of our reaction resulted in a quantification limit of 0.03 bacilli. We then conducted a cross-sectional/cohort-based study of diagnostic accuracy. Patients were included, and skin samples were divided into four layers: epidermis, superior dermis, inferior dermis, and hypodermis. We also quantified M. leprae in nasal swabs of the included patients and compared the results to the number of household contacts also diagnosed with leprosy. One hundred patients with a clinical presentation compatible with leprosy were allocated to the leprosy or control group. Although the parasite load was greater in the superior and inferior dermis, M. leprae DNA was found in all skin layers. The best sensitivity was observed for the superior dermis using the presence of any quantifiable bacillus DNA as the threshold [sensitivity=59.26% (95% CI=45.97-71.32)]. In the epidermis, setting 1 quantifiable bacillus as the threshold resulted in 100% specificity (95% CI=92.29-100). The number of bacilli found in nasal swabs was not significantly related to the number of household contacts also diagnosed with leprosy. Paucibacillary patients tested positive only for bacillus fragments in nasal swabs but not for the entire bacilli. We can conclude that superficial biopsies might result in sensitivity loss, although different skin sample types will have little influence on the final accuracy. In contrast, threshold changes greatly influence these properties. Paucibacillary patients may not be a relevant source of disease transmission.