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

Abstract

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.

Can Leprosy Reaction States Mimic Symptoms of Fibromyalgia? A Cross-Sectional Analytical Study

“…We included patients who received a formal diagnosis of leprosy according to the criteria of the World Health Organization (WHO), who were suspected of experiencing an active reactional state and who had not been diagnosed with fibromyalgia at the beginning of leprosy treatment according to the assessment instruments described elsewhere ( 1 ). The diagnosis of leprosy was always made by a dermatologist based on the criteria of the WHO ( 15 ), and at least one of the following findings was required for diagnosis: (1) skin lesion with altered sensitivity; (2) compatible peripheral neurological alterations, including asymmetric neural thickening, motor deformities, or hypoesthetic areas; (3) parasitological finding of M. leprae on smear examination or skin biopsy (performed for all patients in our center).…”

“…Leprosy is an infectious disease with chronic progression and intense immunological manifestations caused by Mycobacterium leprae and Mycobacterium lepromatosis ( 1 ). The disease mainly affects the skin and peripheral nerves.…”

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

“…The primer pair selected for this study resulted in a 148-base pair product (Table 1). 14,15 Reactions were performed in a final volume of 30 mL containing 1x reaction buffer, 0.2 mM dNTPs, 1.5 mM Waltham, USA), 0.2 mM of each primer (Invitrogen, Waltham, USA), ultrapure water and 50−100 ng of genomic DNA. Amplification was performed with a T100 Thermal Cycler (Bio Rad, Hercules, USA) with an initial denaturation period of 3 min at 94°C followed by 15 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 30 s, followed by 20 cycles of 94°C for 1 min, 56°C for 30 s, and 76°C for 1 min.…”