Development of a combined RLEP/16S rRNA (RT) qPCR assay for the detection of viable M. leprae from nasal swab samples

Abstract

Background Leprosy continues to be a health problem in endemic areas. More than 200,000 new cases of leprosy per year suggest that transmission of the disease is still ongoing, presumably as airborne infection through nasal droplets. Late diagnosis supports continued transmission and increases the individual risk for functional disabilities. Laboratory tools are considered beneficial to facilitate early detection and clinical assessment of cases. The aim of this study was to validate molecular tools allowing detection, quantification and assessment of viability of M. leprae from nasal swab samples which are easy to obtain without the need of any invasive procedures. Methods Validation of two real-time PCRs detecting M. leprae DNA (RLEP qPCR) and RNA (16S rRNA RT qPCR) was conducted on “must not detect”/“must detect” samples and 160 pre-treatment nasal swab samples from 20 clinically diagnosed multibacillary (MB) leprosy patients from Togo. Results Both assays were 100% M. leprae specific and showed analytical sensitivities of three templates each. Out of 20 clinically diagnosed MB leprosy patients, 15 (75.0%) had a positive RLEP qPCR result from nasal swab samples. The 16S rRNA RT qPCR detected viable bacilli in nasal swab samples of ten out of these 15 RLEP positive patients (66.7%). Conclusion The combined RLEP/16S rRNA (RT) qPCR assay provides a sensitive and specific tool to determine the bacterial load and viability of M. leprae from nasal swab samples and is applicable for early diagnosis, monitoring treatment response and investigating the role of nasal carriage of M. leprae in human-to-human transmission through aerosol infection. Electronic supplementary material The online version of this article (10.1186/s12879-019-4349-9) contains supplementary material, which is available to authorized users.

Ultrasensitive Detection of Bacillus anthracis by Real-Time PCR Targeting a Polymorphism in Multi-Copy 16S rRNA Genes and Their Transcripts

“…While these numbers are well in agreement, somewhat lower numbers of 9 × 10 3 ribosomes have been determined for exponentially growing cells of Bacillus subtilis [18]. While unexplored for B. anthracis, bacterial detection using rRNA genes and transcripts has been successfully harnessed to challenge previous limits of detection (LoD) for other pathogens [19][20][21].…”

“…Ribosomal RNA detection was also utilized for Mycobacterium leprae diagnosis by the same research team. Here, an LoD of three M. leprae target copies was achieved for a novel 16S rRNA RT-PCR assay; the same value as determined for the M. leprae specific multi-copy repetitive DNA target assayed in parallel [21]. At first glance, these values do not especially speak in favor of querying for 16S rRNA transcripts; however, one has to consider the high numbers of these molecules per cell in comparison to DNA markers (including the high-copy ones).…”

Report on an unusual case of leprosy from Germany: just an exception of the rule?

“…This time confirmed by histopathology of a skin biopsy of a macular lesion, Ziehl-Neelsen microscopy and RLEP qPCR (repetitive element RLEP of the M. leprae genome) of a skin biopsy and nasal swab samples, as well as an unaltered high anti-PGL-I antibody titer. The bacillary load of the patient as determined by microscopy (nasal swab: BI 4+) and quantification by RLEP qPCR (skin biopsy: ~ 300,000 bacteria in 100 µl extract, nasal swab: ~110,000 bacteria in 100 µl extract) was remarkably high [14]. Molecular drug resistance testing at the Global Health Institute of the Ecole Polytechnique Fédérale de Lausanne, Switzerland, was conducted according to standardized methods used by the WHO surveillance network for antimicrobial resistance in leprosy and revealed no resistance to rifampicin, dapsone or ofloxacin [15,16].…”

“…However, the reliability of the MI is limited as multiplication of M. leprae from non-solid organisms has been reported [17,18]. A molecular viability assay (16S rRNA RT qPCR [14,19]; supplementary material 1) proved the presence of viable M. leprae in nasal swab samples. Subsequently the patient was put on a second course of MDT (due to delay in delivery the medication started with two doses of rifampicin and MDT was given with 11 days delay).…”

High rate of Mycobacterium leprae infection and nasal colonization among household contacts of previously undiagnosed leprosy patients

“…2 Thus, the disease continues to be an important public health challenge in leprosy endemic areas. 1,3 Most people residing in a leprosy-endemic setting have been exposed to M. leprae but few develop the disease. 4 Solid evidence exists of an increased risk to individuals living in close contact with leprosy patients.…”