Bacilloscopy and polymerase chain reaction of slit-skin smears and anti-phenolic glycolipid-I serology for Hansen’s disease diagnosis

Abstract

The bacilloscopy of the slit-skin smear (SSS) is the exclusive laboratory test associated with dermato-neurological evaluation for Hansen’s disease (HD) diagnosis; however, it is negative in the majority of PB or primary neural forms. Thus, a PCR technique involving different sequences and target genes has been performed with an aim to increase the sensitivity and specificity of M. leprae identification, especially in patients with low bacillary loads. Additionally, serological assays based on antibody response reflect infection levels and indicate that this could be a simpler, less invasive technique for estimating M. leprae exposure. Serological tests and PCR have been shown to be more sensitive and accurate than the SSS. Our study aimed to measure accuracy and performance among the SSS and PCR of dermal scrapings stored on filter paper and APGL-I serology for diagnosis in HD. A cross-sectional study analyzing the medical records (n = 345) of an HD outpatient-dermatology clinic from 2014 to 2021 was conducted. Accuracy performance parameters, correlation, and concordance were used to assess the value among the SSS, PCR, and APGL-I exams in HD. The SSS presented 24.5% sensitivity, 100% specificity, 37.4% accuracy, and the lowest negative predictive value (21.5%). The PCR assay had 41, 100, and 51% sensitivity, specificity, and accuracy, respectively. PCR and APGL-I serology increased the detection of HD cases by 16 and 20.6%, respectively. PCR was positive in 51.3% of patients when the SSS was negative. The SSS obtained moderate concordance with PCR [k-value: 0.43 (CI: 0.33–0.55)] and APGL-I [k-value: 0.41 (CI: 0.31–0.53)]. A moderate positive correlation was found between the APGL-I index and the bacillary index (r = 0.53; P < 0.0001). Thus, the use of the SSS is a low sensitivity and accuracy method due to its low performance in HD detection. The use of PCR and serological tests allows for a more sensitive and accurate diagnosis of patients.

Serological testing for Hansen’s disease diagnosis: Clinical significance and performance of IgA, IgM, and IgG antibodies against Mce1A protein

“…Indirect ELISA was used to measure the α-PGL-I IgM titer of every serum sample and the cut-off was based on the OD average among healthy subjects multiplied by 2.1 plus 10%, according to a previously reported protocol ( 7 , 12 , 15 ). Serology was performed with an ND-O-BSA (PGL-I)-based glycoconjugate of bovine serum albumin (NR-19346.…”

“…Total DNA extraction from a skin biopsy and/or earlobes and at least one elbow, knee and/or lesion slit-skin smear sample was performed with the QIAamp DNA Mini Kit (Qiagen, Germantown, MD, cat: 51306) according to the manufacturer’s protocol. DNA was used to perform quantitative PCR-RLEP according to a previously reported protocol ( 7 , 16 ). The quantitative PCR (qPCR) result was considered positive for the detection of M. leprae DNA with amplification up to a 40.0 cycle threshold (Ct) and melting temperature at 87.5°C.…”

“…Quantitative evaluation of IgA, IgM, and IgG antibody α-Mce1A protein was performed by indirect ELISA according to a previously reported protocol ( 7 , 11 , 12 ). Purified recombinant Mce1A protein was provided by Dr. LW Riley (University of California, Berkeley, CA, USA).…”

“…The cut-off point was based on mean OD between healthy controls compared to samples from patients with HD. The OD data were analyzed by receiver operating characteristic (ROC) curves to determine the cut-off point highest and matched sensitivity, specificity, and likelihood ratio, as previously described ( 7 , 11 , 12 ). For all assays, negative control samples from healthy individuals with no history of diagnosis or contact with HD, positive samples for α-Mce1A antibodies from patients diagnosed with HD, and wells considered blank without the addition of specific antibodies and with peroxidase-linked second antibody for each immunoglobulin tested were added.…”

“…More recently, the introduction of molecular biology to identify bacillus DNA in clinical samples (skin, nasal swab, and intradermal scraping) has increased the probability of detecting new cases while maintaining high specificity and has shown that the polymerase chain reaction (PCR) may be used to confirm most field cases ( 8 ). On the other hand, PCR is an expensive method not available to all laboratories for the diagnosis of HD, in addition to the absence of a gold standard laboratory test ( 7 ).…”