Diagnostic research: Neglected mycobacterial diseases

Project leader: Dr. med. Marcus Beißner

Buruli ulcer (BU), the third most common mycobacterial disease after tuberculosis and leprosy, has so far been reported from 35 countries worldwide, with the focus of the disease being in West and Central Africa. BU is a disease of the skin and subcutaneous fatty tissue; bone involvement is also possible. Scarring and contractures can lead to severe disabilities, so early diagnosis and treatment are essential. Treatment involves eight weeks of antimycobacterial combination therapy with rifampicin and clarithromycin, possibly in combination with surgical measures. The WHO requires laboratory confirmation of 70% of all BU cases worldwide. This is preferably carried out using PCR, a highly sensitive and specific diagnostic method, whereby an insertion element (IS2404) present in >200 copies in the bacterial genome is detected. However, the use of PCR is restricted to appropriately equipped reference laboratories. In order to improve access to diagnostics for the affected population, most of whom live in rural areas, a panel of experts from the WHO and the Foundation for Innovative Diagnostics (FIND) discussed the introduction of field-ready point-of-care diagnostic methods in decentralized laboratories in endemic countries. One promising technology on the WHO and FIND agenda is loop-mediated isothermal amplification (LAMP), a simple, robust and fast method that does not require highly specialized laboratory equipment. In collaboration with FIND, the Noguchi Memorial Institute for Medical Research (NMIMR), Accra, Ghana, and the company AmplexDiagnostics, Dr. Beißner's team developed, optimized and validated the prototype of a dry reagent-based (DRB-) IS2404 LAMP, which can be carried out using a portable fluorimeter. In a follow-up project, the performance characteristics of the assay and its suitability for decentralized laboratories are now to be tested in a field study in Togo. For this purpose, diagnostic samples from 187 suspected BU cases will be tested simultaneously in a decentralized laboratory and in the Togolese BU reference laboratory using IS2404-DRB-LAMP and IS2404 real time qPCR (the standard test method used in Togo). If the IS2404-DRB-LAMP proves successful under field conditions, the assay will be used routinely in decentralized laboratories in West Africa. A comparable study is currently being conducted by our cooperation partners in Ghana.

Project leader: Malkin Saar, MSc

According to the Ridley-Jopling classification, which is based among other things on bacteriological findings, leprosy is divided into tuberculoid, borderline and lepromatous forms. According to a simplified, field-based WHO classification, a distinction is made between paucibacillary forms (≤5 lesions) and multibacillary forms (>5 lesions) based solely on the number of lesions, which are treated differently. However, without bacteriological evidence, this simplified classification can lead to incorrect classification and consequently to inadequate treatment. Various studies have evaluated the bacteriological detection of M. leprae from skin biopsies, skin smears and nasal swabs using different PCR-based methods for the diagnosis and classification of the disease. Despite the clear superiority of PCR over other diagnostic methods, the use of PCR is currently not recommended by the WHO due to the complexity of the method, the need for trained personnel and the lack of standardized, commercial test kits. LAMP technology would provide a remedy here. Based on the experience with the dry reagent-based Buruli-DRB-LAMP described above, our research group developed and validated a leprosy DRB-LAMP assay for the detection of the leprosy-specific repetitive element RLEP from nasal swabs. Clinical validation of the assay is currently being carried out using patient samples from Togo.

Project leader: Dr. med. Marcus Beißner

Doctoral students: Anna Wöstemeier (completed), Malkin Saar, MSc (ongoing)

More than 200,000 new infections per year indicate that transmission of leprosy is still ongoing, with aerogenic transmission via droplet infection via the oral and nasal mucosa being assumed. The working group developed and validated a combined RLEP real time qPCR/16 S rRNA RT qPCR assay (detection and quantification of M. leprae DNA/detection of mycobacterial RNA≙viability) to detect, quantify and confirm the viability of M. leprae in the nasal mucosa of patients and their contacts. The sensitive and specific assay is used in patients at our outpatient clinic for early laboratory confirmation of suspected cases and to monitor the success of treatment and also allows epidemiological investigations into the transmission of the pathogen.

A study in Togo was designed to determine the proportion of infected persons among contact persons of leprosy patients and to clarify the conditions under which transmission occurs. Initially, nasal swabs from 80 suspected leprosy cases were examined using the combined assay. 52 cases (65%) were laboratory-confirmed, 24 of 46 laboratory-confirmed cases (52%) had a positive viability test.

Of 36 laboratory-confirmed patients recruited as index patients (68% of whom had positive evidence of viability), 255 close contacts (mostly household contacts, up to ten per index patient) were recruited and also tested using the combined assay. In 11% (n=27) of all contact persons, M. leprae DNA was detected by nasal swab (all positively tested contact persons came from the environment of only 14 of the 36 index patients), one contact person also had a positive viability test (0.39% of all contact persons).

Index patients with a high bacterial load (>10,000 bacteria/nasal swab) and viable bacteria in the nasal swab generally had more positive contact persons than index patients with a lower bacterial load and no evidence of viable bacteria. Similarly, families with several cases of leprosy had more positive contacts than families with only one case of the disease.

Project partners: Prof. Dr. Gisela Bretzel, Dr. Marcus Beißner

In view of the lack of standardization of molecular biological diagnostic methods and decreasing numbers of laboratory-confirmed Buruli ulcer cases, the BU-Labnet, an association of 11 PCR diagnostic laboratories from 9 African countries, was founded in October 2019 at a meeting at the Institute Pasteur, Cameroon, under the auspices of the WHO. The aim of the network is to create uniform quality standards by means of standardized methods, reagents and laboratory materials, as well as internal and external quality control through round robin tests and laboratory supervision in conjunction with training measures. In the medium term, the network offers prospects for accreditation of the participating laboratories as well as a platform for international research cooperation in the field of Buruli ulcer and other neglected tropical diseases.