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

Leprosy caused by Mycobacterium leprae is a neglected, chronic infectious disease predominantly affecting skin and peripheral nerves. More than 200,000 new cases are detected annually and up to 60% of patients have peripheral nerve damage at diagnosis and are therefore prone to long-term morbidity. Environmental sources may be involved in the ongoing dissemination of M. leprae, but aerosol spread through the upper respiratory tract is considered the principal means of transmission. The disease is spectral and categorized according to the Ridley & Jopling classification [1]. Patients with a strong cell-mediated immune response have few lesions with few or no detectable mycobacteria (tuberculoid forms), whereas patients anergic to M. leprae have multiple lesions with numerous mycobacteria (lepromatous forms). Between these two poles borderline forms exist. To guide treatment decisions, WHO has introduced a simplified classification based on the number of lesions. Whereas previously paucibacillary (PB) cases (up to five skin lesions) were treated for six months with rifampicin and dapsone, and multibacillary (MB) cases (more than five skin lesions) for 12 months with rifampicin, dapsone and [2] clofazimine, current WHO recommendations valid since August 2018 envisage to treat PB patients for six months and MB patients for 12 months with rifampicin, dapsone and clofazimine (multi-drug therapy, MDT). The diagnosis of leprosy is clinical. However, an estimated 30% of patients, including many MB cases, do not present conclusive clinical signs and classification of patients based on counting of lesions alone is subject to error. Therefore, application of auxiliary laboratory based tools is considered beneficial to support clinical diagnosis and classification. Well-established procedures are histological diagnosis as well as determination of the bacteriological index (BI, representing the quantitative bacillary load) and the morphological index (MI, representing the percentage of intact solid stained and presumably viable bacilli) by means of examination of Ziehl-Neelsen stained slit skin smears (SSS). Furthermore, phenolic glycolipid I serology is considered an excellent surrogate marker for the bacterial load and can aid in clinical management such as patient classification and monitoring of treatment [3‐10].

Considerable progress has been made in the field of molecular diagnostics. PCR techniques have been applied to investigate possible environmental sources for dissemination of M. leprae as well as the aerosol route of infection by means of nasal carriage [11‐13]. Regarding diagnostics, quantitative polymerase chain reaction (qPCR) technology is considered at least 20 times more sensitive than microscopic detection and becomes increasingly important for early diagnosis and for difficult-to-diagnose cases, such as patients with negative microscopy, pure neural leprosy, or differential diagnosis of lesions with inconclusive histopathology [14]. Although the diagnostic sensitivity of (q) PCR assays is considered highest for skin biopsies, M. leprae DNA detection rates of more than 80% from SSS and nasal swab samples from clinically suspected MB cases, as well as 30–40% from SSS and nasal samples of BI negative PB cases were reported. Among a range of possible gene targets, the M. leprae specific repetitive element RLEP with an amplifiable copy number varying between 19 and 37 according to mutations in the primer binding sites, has been identified as the most suitable target for diagnostic applications [14‐20].

As diagnostic PCRs only amplify M. leprae DNA and the pathogen cannot be cultured in-vitro, alternative technologies are required to determine mycobacterial viability. Molecular viability assays targeting ribosomal (e.g. 16S rRNA) or messenger RNAs - only detectable from viable/replicating bacteria – are available for monitoring treatment of patients with tuberculosis and Buruli ulcer. Comparable viability assays were developed for M. leprae and are applicable for environmental studies and assessment of treatment response in leprosy patients [11, 12, 14, 21‐23].

Mainly attributable to the introduction of MDT in the 1980s and its widespread free of charge distribution since 1995 through WHO, the leprosy elimination goal (i.e. < 1 patient per 10,000 population) was achieved in most countries in 2000. However, worldwide more than 200,000 new cases are still reported each year indicating ongoing transmission of the disease. In particular late diagnosis supports continued transmission and increases the individual risk for functional disabilities [7, 30]. Among the available diagnostic techniques applicable for laboratory confirmation, RLEP qPCR has shown the highest specificity and sensitivity for the detection of M. leprae in clinical samples. A range of studies validated the performance of RLEP qPCR on skin biopsies and SSS. Validation data for nasal swabs, a less invasive sampling technique which is applicable in the field, are however scarce. In addition, only a few studies applied an RLEP assay covering the complete range of the known RLEP variants, RLEP 1–4 [13, 14, 17, 20, 27, 31]. Therefore, the aim of this study was to validate an RLEP qPCR targeting the entire set of RLEP sequences applicable for nasal swabs.

Technical validation revealed a RLEP qPCR specificity of 100%, a very high analytical sensitivity of 0.1 M. leprae genome equivalents, an efficiency of 95.6% and low intra- and inter-assay variabilities. The RLEP qPCR positivity rate of 75% falls within the range of data reported from other studies applying RLEP qPCR for the detection of M. leprae DNA from nasal swab samples (47–75% [13, 31]), SSS (83% [20]) and skin biopsies (87% [17]) from clinically classified MB patients.

Beside early identification of clinical leprosy among RLEP qPCR positive contacts, one possible application of RLEP qPCR consists in monitoring the decrease of the bacillary load during MDT. However, M. leprae DNA is detectable for at least two years after MDT and mere quantification of bacilli does not provide definite information on the viability of possible remaining organisms. As M. leprae cannot be cultured on artificial media, proof of viability requires more sophisticated tools. Detection of M. leprae RNA is considered a suitable alternative to identify viable/replicating organisms, and a few studies demonstrated the applicability of RNA assays for assessment of bacterial viability under MDT in SSS and skin biopsies [14, 21, 22]. RNA assays have also gained an important role in transmission studies with viable M. leprae being detected in environmental samples derived from the immediate vicinity of houses of leprosy patients [11, 12]. To our knowledge application of RNA assays to investigate human-to-human transmission is still pending.

In our setting, a viability assay should serve two purposes. First, there was the need for a diagnostic tool to monitor the treatment response of leprosy patients attending our outpatient clinic in Munich, Germany. Furthermore, an assay facilitating transmission studies in research settings in Africa was required. As already shown by Martinez et al. analysis of M. leprae mRNA (e.g. sodA) to predict viability of the bacilli is limited to short-term experimental settings due to a low sensitivity in clinical samples [22]. The low sensitivity for mRNA detection from clinical samples in general was also described for other (myco-) bacterial pathogens [32], but some authors argued that rRNA – though highly sensitive – may also be detected from dead bacteria (among these also metabolic active but culture negative bacilli, e.g. MTBC) [32‐34]. However, findings of rRNA analyses obtained from other pathogens may not be one-to-one transferred to M. leprae due to the genus- and even species-specific ribosomal transcriptome [35] and RNA applications for M. leprae should follow the existing knowledge on this specific species. For M. leprae Prakoeswa et al. recently proofed the findings of an earlier study by Haile et al. that 16S rRNA is rapidly degraded in dead M. leprae and can thus be used as marker of viability [36, 37]. Therefore, based on the 16S rRNA RT qPCR first described by Martinez et al. [22], a viability assay for non-invasive nasal swab samples - facilitating repeated sampling as well as sampling under field conditions - was designed. Technical validation revealed a high analytical sensitivity of three M. leprae genome equivalents, qPCR efficiency of 97.8% and low intra- and inter-assay variabilities. The 16S rRNA positivity rate among RLEP positive patients was 66.7% (10/15). Among the five patients without detectable viable M. leprae, one was probably subject to sampling error as GAPDH mRNA RT qPCR was also negative.

The present study constitutes the first application of an RNA based viability assay for M. leprae on nasal swabs. The RNA assay in combination with quantification by RLEP qPCR was successfully tested for monitoring the treatment response in two MB patients from Germany. Clinical samples of the Togolese validation cohort were not bacteriologically confirmed as neither microscopic assessment of skin smears nor histopathological analysis of skin biopsies was part of the routine diagnostic procedure in Togo. However, for one of our German patients we had the opportunity to extensively analyze corresponding nasal swab samples by microscopy and the two molecular assays. A correlation was found between the bacillary load as determined by RLEP qPCR and BI, as well as the presence of viable bacilli as detected by the RNA assay and MI [38]. It must be noted that the viability assay developed by our group does not allow quantification of RNA as gene expression studies were out of the scope of this work.

Meanwhile investigations on human-to-human transmission and nasal carriage of viable M. leprae among untreated MB patients and contact persons in Togo are underway [unpublished data]. Whereas the application of the molecular assays presented no challenges in our German laboratory, several limitations may hamper the realization of its full potential in resource poor settings. The assays in their current format are costly and need adequate infrastructure. Some reagents require expensive dry-ice shipping and continuous stocking of the reagents in Togo means at least three shipments per year. This in turn implies the need for a well-established laboratory management system, which in Togo has been provided by the accredited laboratory of INH [39, 40]. Whereas in Germany the follow-up of leprosy patients can be done in close intervals as needed, in endemic regions beside test costs also the significant mobility of populations in endemic regions may impede close routine follow-up.

In order to provide laboratory based management and follow-up of leprosy patients attending our outpatient clinic in Munich, Germany, as well as to facilitate transmission studies in Africa, a combined RLEP/16S rRNA (RT) qPCR assay was developed. The 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. Whereas in our own setting fortunately neither financial nor logistical restraints hampered the application of the assay on individual patients, for the most part these assays may not be applicable for individualized medicine but rather for epidemiological research issues.