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Link to original content: https://pubmed.ncbi.nlm.nih.gov/27888674/
qPCR detection of Mycobacterium leprae in biopsies and slit skin smear of different leprosy clinical forms - PubMed Skip to main page content
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. 2017 Jan-Feb;21(1):71-78.
doi: 10.1016/j.bjid.2016.09.017. Epub 2016 Nov 24.

qPCR detection of Mycobacterium leprae in biopsies and slit skin smear of different leprosy clinical forms

Michelle de Campos Soriani Azevedo  1 , Natália Mortari Ramuno  2 , Luciana Raquel Vincenzi Fachin  2 , Mônica Tassa  2 , Patrícia Sammarco Rosa  2 , Andrea de Faria Fernandes Belone  2 , Suzana Madeira Diório  2 , Cleverson Teixeira Soares  2 , Gustavo Pompermaier Garlet  1 , Ana Paula Favaro Trombone  3
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qPCR detection of Mycobacterium leprae in biopsies and slit skin smear of different leprosy clinical forms

Michelle de Campos Soriani Azevedo et al. Braz J Infect Dis. 2017 Jan-Feb.

Abstract

Leprosy, whose etiological agent is Mycobacterium leprae, is a chronic infectious disease that mainly affects the skin and peripheral nervous system. The diagnosis of leprosy is based on clinical evaluation, whereas histopathological analysis and bacilloscopy are complementary diagnostic tools. Quantitative PCR (qPCR), a current useful tool for diagnosis of infectious diseases, has been used to detect several pathogens including Mycobacterium leprae. The validation of this technique in a robust set of samples comprising the different clinical forms of leprosy is still necessary. Thus, in this study samples from 126 skin biopsies (collected from patients on all clinical forms and reactional states of leprosy) and 25 slit skin smear of leprosy patients were comparatively analyzed by qPCR (performed with primers for the RLEP region of M. leprae DNA) and routine bacilloscopy performed in histological sections or in slit skin smear. Considering clinical diagnostic as the gold standard, 84.9% of the leprosy patients were qPCR positive in skin biopsies, resulting in 84.92% sensitivity, with 84.92 and 61.22% positive (PPV) and negative (NPV) predictive values, respectively. Concerning bacilloscopy of histological sections (BI/H), the sensitivity was 80.15% and the PPV and NPV were 80.15 and 44.44%, respectively. The concordance between qPCR and BI/H was 87.30%. Regarding the slit skin smear, 84% of the samples tested positive in the qPCR. Additionally, qPCR showed 100% specificity, since all samples from different mycobacteria, from healthy individuals, and from other granulomatous diseases presented negative results. In conclusion, the qPCR technique for detection of M. leprae using RLEP primers proved to be specific and sensitive, and qPCR can be used as a complementary test to diagnose leprosy irrespective of the clinical form of disease.

Keywords: Bacilloscopy; Leprosy; Mycobacterium leprae; qPCR.

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Figures

Fig. 1
Fig. 1
Quantitative PCR standard curve. A standard curve was prepared from the purified DNA of M. leprae. It was performed by serial dilution (1:10) with the initial and final points 0.3 ng and 3 fg, respectively, and the limit of detection was equal to 300 fg.
Fig. 2
Fig. 2
Detection of M. leprae in biopsy samples by qPCR technique. Data reported in scatter dot plot as medians, excluding negative results. (A) Patients divided in seven groups (n = 107). (B) Patients grouped as paucibacillary (BI/H ≤ 1+) and multibacillary (BI/H ≥ 2+) forms, excluding negative results (n = 107). BI/H: bacilloscopy of histological sections, TT: tuberculoid, BT: borderline-tuberculoid, BB: borderline-borderline, BL: borderline-lepromatous, LL: lepromatous, RR: reversal reaction, ENL: erythema nodosum leprosum. #p < 0.05.
Fig. 3
Fig. 3
Detection of M. leprae in slit skin smear samples by qPCR technique. Data reported in scatter dot plot as medians, excluding negative results (n = 21). Samples grouped according to bacilloscopy of slit skin smears (BI/S).
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