• Grant: LRI Regular Grant
• Research priorities: Diagnostic tests
• Country: Bangladesh
• Project no.: 703.15.07
• Budget: € 1,495,000
• Duration: January 2015 - March 2019
• Status: Completed
• Co-funding partners: Turing Foundation

Full project title:
Field evaluation of novel immunodiagnostic tools for early detection of leprosy in a BCG vaccination field trial amongst contacts of leprosy patients (INDIGO)

Project coordination
Erasmus MC, University Medical Center Rotterdam (The Netherlands)

Partners
Leiden University Medical Center (The Netherlands)
The Leprosy Mission International (Bangladesh)

Aim: This study aimed to identify compounds of the immune system that are characteristic for the occurrence of leprosy and may differentiate between persons affected by leprosy, their contacts and healthy controls. Such compounds are called leprosy-specific biomarkers. 

Final project summary
Although a combination of antibiotics (multidrug therapy) is very effective at curing clinical leprosy, it is insufficient to reduce transmission of M. leprae, the causative agent of leprosy, in endemic populations as witnessed by stable new case detection rates in many leprosy endemic countries. It is therefore important to identify risk factors, transmission patterns and preventive measures that may be used as tools for early detection and prevention of leprosy. The aim of the study was to understand in more detail how people respond immunologically to the presence of M. leprae, in order to obtain insight into which immunological responses in people indicate that they are developing leprosy disease. In addition, since the BCG vaccine can induce protection against leprosy, BCG vaccination of contacts of leprosy patients will allow identification of immune responses that reflect protection against leprosy.

This was a long-term project to test many leprosy patients, their contacts, and people from the general population in Bangladesh on the presence of infection with M. leprae and the effect of BCG vaccination and treatment with an antibiotic (rifampicin) of contacts on host biomarker profiles. In a separate (non-LRI funded) project, field-friendly tests were developed to detect leprosy disease and M. leprae infection in endemic areas. This field-tests were based on differences in immune- biomarkers between those who have or developed disease and healthy contacts (exposed but remain healthy) and controls from the same area. Early diagnosis followed by (prophylactic) treatment of people who will otherwise develop leprosy will prevent transmission of the bacteria and possibly life-long disabilities in many of these people.

The researchers have identified several compounds in blood that were present in different levels in people with leprosy and contacts thereof likely infected with the bacterium compared to people who did not have any known contact with leprosy patients.

Among contacts receiving BCG and single dose rifampicine (SDR), one third of new cases were detected in the window between BCG vaccinatino and SDR. Therefore,  BCG vaccination followed by single dose rifampicine (SDR) cannot be recommended as routine intervention in leprosy control. Alternatively, focus on field trials of new candidate leprosy vaccines is a possibility. However, the research group advises contact surveys followed by SDR to eligible contacts of new leprosy cases - in line with tje recent recommendation by the World Health Organisation (WHO).

With respect to the differences in serum proteins between the test groups in this study, several new biomarkers were detected in stimulated whole blood that can discriminate MB and PB leprosy patients from contacts and healthy controls. Importantly, leprosy-associated biomarkers were identified in plasma (i.e. without overnight stimulation), offering potential for application in fingerstick blood-based tests for low resource settings.

Impact

Van Hooij, A, EM Tjon Kon Fat, R Richardus, SJF van den Eeden, Louis Wilson, CJ de Dood, R Faber, K Alam, JH Richardus, PLAM Corstjens and A Geluk. 2016. Quantitative lateral flow strip assays as user-friendly tools to detect biomarker profiles for leprosy. Sci. Rep. 6:34260;doi:10.1038/srep34260

Van Hooij, A, EM Tjon Kon Fat, SJF van den Eeden, L Wilson, M Batista da Silva, CG Salgado, JS Spencer, PLAM Corstjens, and A Geluk. 2017. Field-friendly serological tests for determination of M. leprae-specific antibodies. Scientific Reports 2017;7(1):8868.

Richardus, RA, K van der Zwet, A van Hooij, L Wilson, L Oskam, R Faber, SJF van den Eeden, D Pahan, K Alam, JH Richardus and A Geluk. 2017. Longitudinal assessment of anti-PGL-I serology in contacts of leprosy patients in Bangladesh. PLoS NTDs 2017, 11(12):e0006083.

Richardus RA, van Hooij A, van den Eeden SJF, Wilson L, Alam K, Richardus JH, Geluk A. 2018. BCG and Adverse Events in the Context of Leprosy. Front Immunol. 2018;9:629.

Blok DJ, SJ de Vlas, A Geluk, JH Richardus. 2018. Minimum requirements and optimal testing strategies of a diagnostic test for leprosy as a tool towards zero transmission: A modelling study. PLoS Negl Trop Dis 12(5): e0006529.

Geluk A. 2018. Correlates of immune exacerbations in leprosy. Seminars in Immunology. 39:111-8.

Van Hooij, A, EM Tjon Kon Fat, M Batista da Silva, R Carvalho Bouth, ACC Messias, AR Gobbo, T Lema, K Bobosha, J Li, X Weng, CG Salgado, JS Spencer, PLAM Corstjens, and A Geluk. 2018. Evaluation of immunodiagnostic tests for leprosy in Brazil, China and Ethiopia. Scientific Reports 8:17920.

Tio-Coma M, Wijnands T, Pierneef L, Schilling AK, Alam K, Roy JC, Faber WR, Menke H, Pieters T, Stevenson K et al. 2019. Detection of Mycobacterium leprae DNA in soil: multiple needles in the haystack. Scientific reports. 9(1):3165.

Corstjens P, van Hooij A, Tjon Kon Fat EM, Alam K, Vrolijk LB, Dlamini S, da Silva MB, Spencer JS, Salgado CG, Richardus JH, van Hees CLM, Geluk A. 2019. Fingerstick test quantifying humoral and cellular biomarkers indicative for M. leprae infection. Clinical biochemistry, Volume 66, pp. 76-82.

Richardus RA, Alam K, Kundu K, Chandra Roy, J, Jafar T, Chowdhury AS, Kahn D, Nieboer D, Faber R, Butlin CR, Geluk A, Richardus JH. Effectiveness of single dose rifampicin given after BCG vaccination in preventing leprosy in close contacts of patients with newly diagnosed leprosy: a cluster randomized controlled trial. 

Presentations: 
- Quantitative lateral flow assay for detection of leprae infection using fingerstick blood. EDCTP forum, Lisbon, Portugal. 18^th September 
    2018.
- Laboratory aspects (training, quality control, consumables, procedure harmonization. Kick-off meeting EDCTP PEOPLE project. Antwerp,
   Belgium. 2^nd October 2018 Title: POC test for childhood TB. NIH Pediatrics TB annual meeting. Cape Town, South Africa. 6th November
   2018.
- Host Biomarker-based POC tests for Leprosy Diagnosis in Hyperendemic Regions. Brazilian Leprosy Congress 2018. Palmas, Brazil. 17^th
   November, 2018.
- High tech at low complexity: development of POC tests for Leprosy & TB. Hôpital Lariboisière, Paris, France. 10^th December, 2018.

• Grant: LRI Regular Grant
• Research priorities: Diagnostic tests
• Country: Philippines
• Project no.: 703.15.05
• Budget: € 390,000
• Duration: May 2015 - September 2019
• Status: Completed
• Co-funding partners: Turing Foundation

Full project title:
Integration of rapid diagnostic tests to facilitate case management of leprosy

Project coordination
Infectious Disease Research Institute 

Partners
Cebu Skin Clinic/ Leonard Wood Memorial Center for Leprosy Research (The Philippines)

Aim: This study evaluated if certain antibody tests could serve as a simple, quantifiable and robust measurement to facilitate referral for expert clinical exam.

Final project summary
This project aimed to rapidly and simply detect and monitor levels of anti-leprosy antibodies in individuals at the greatest risk of developing leprosy to provide an earlier indication of M. leprae infection/ progression to leprosy. The researchers  hypothesised that this data could be used to supplement, or potentially replace, clinical exams as the initial entry point to expert leprosy diagnosis, care and management. As a secondary aim,  these responses were assessed in patients undergoing multi-drug therapy to evaluate their ability to identify relative risk of treatment complications. 

Acceptance of blood testing was high at intake both surveillance and monitoring and remained high for the 4 year duration of the project. Indeed, 89.2% of the patient population with strongest indication of ‘definitely’ to the query of “If a study was looking to use a rapid and simple diagnostic and treatment monitoring tool for leprosy, would you be interested in participating?” at the start of the study, although this waned in the fourth year down to 59.2%. This was supported by an overwhelming majority of patients being willing to submit to RDT evaluations. Even as enthusiasm among patients reduced in the end stages of the study, it was only slightly as 32.5% still responded that they would ‘probably’ participate.  To determine if the use of RDT would be viewed favourably or unfavourably, newly diagnosed leprosy patients were asked if they thought it would be beneficial to have a simple and rapid diagnostic and treatment monitoring test for leprosy. At the time of diagnosis 94.0% responded ‘yes’ with the remaining 6.0% expressing some hesitancy by responding ‘possibly’. Of note, however, none of the patients responded negatively to the potential use of RDT and over time there was a gradual strengthening of the responses. By 4 years after diagnosis 98.3% patients were responding ‘yes’ while the other 1.7% responded with ‘possibly’ beneficial.

As another measure of acceptance the researchers queried patients as to their willingness to have contacts approached for potential testing. During the initial questioning one fifth (20.8%) of patients were not in favor of using RDT to monitor their contacts, but by the end of the study this had reduced to only 1.7% responding negatively to an expansion of testing among their contacts. When a variety of contact populations were considered, patients indicated that testing among household members was preferred over broader evaluations into the community. With the conversion of responses from no testing to acceptance of testing in contacts, the proportion of patients that indicated a preference for testing among their household members increased over time. When asked about the frequency of testing, the most common response was ‘anytime as needed’ and this response become more popular as the study progressed. With this, the proportion of patients that preferred regular testing among their contacts at either a monthly or 6 monthly interval became less prevalent.

To determine if the use of RDT would be viewed favorably or unfavorably beyond the patient group, HC were asked how they perceived a simple and rapid diagnostic tool for monitoring of exposure/ possible exposure to M. leprae. At each of the times they were questioned over 99% of HC responded affirmatively (combination of ‘yes’ and ‘possibly’) that they thought it would be beneficial, with 97.7% and more saying that they would like such a tool to be used in their household/ community. While the most common response to preferred frequency was ‘anytime as needed’, there was an interesting transition with annual testing become more preferred than testing every 6 months by the end of the study.

Almost universally HC thought that such a test would be useful for controlling leprosy and its complications with the perception among the vast majority being that it was ‘very important’. To directly address acceptability, we asked HC if they would personally be willing to participate in testing. While 86.2 and 84.5% of the total and selected for follow up HC groups responded positively (‘probably’ and ‘definitely’) , respectively, 59.2% of the 600 HC interviewed at intake responding that they would definitely be willing to participate, there was a modest enrichment to 68.4% responding similarly among the HC selected for routine questioning. By the conclusion of the study 92.1% of HC queried repeatedly indicated that they would definitely participate in testing. Taken together, this information indicates that the use, or at least the examination of use, of a RDT for leprosy is acceptable within this HC population and furthermore, this acceptability rose over time.

Among the total initial intake of 200 patients, 15 presented with reactional episodes at the time of diagnosis (6 ENL, 9 RR). Among the 130 patients that were subsequently monitored 8 patients presented with reactional episodes (4 ENL, 4 RR) during their initial diagnosis and a total of 69 (53.1%) presented with, or developed, at least one reactional episode in the 48 months following the initiation of MDT. 42 patients had type 1 reversal reactions, 26 displayed ENL, and 1 patient had both types of reaction. It was found that 44 of 69 had their first reactional episodes while undergoing MDT (months 1-12), while 25 of the patients had symptoms of reactions at one observation time. The majority (44 of 69; 63.8%) experienced either multiple episodes or persistent chronic reactions that spanned multiple assessment periods.

Leprosy Detect^TM fast ELISA revealed that, as a group, patients that developed ENL at some point in time generally had a significantly higher mean antibody response at the time of diagnosis than patients that did not develop reactions (p-value = 0.0147). Patients that developed reversal reactions at some point in time had antibody response at the time of diagnosis that were similar to patients that did not develop reactions but significantly lower than those of future ENL patients (p-values = 0.5675 and 0.0012, respectively). The difference between ENL patients and patients that did not experience reactions was not, however, observed using RDT at intake.

For patients with the subsequent development of reactional episodes, however, there was no clear difference in the profile of the response relative to patients that did not have reactional episodes. For the group of patients that did not develop reactions it took 9 months for Leprosy Detect^TM fast ELISA results to become statistically significantly reduced from the intake values, whereas for patients in the groups that experienced either RR or ENL this took 6 months. Surprisingly, the kinetics of reduction of signal in OnSite Leprosy Ab Rapid Test were markedly different, with the time taken to produce a statistically significantly reduction relative to intake values being 6 months patients that did not develop reactions, 18 months for patients that developed RR and only 3 months for patients that would develop ENL. These results indicate an intriguing variance between ELISA and RDT that likely arises due to the combination of the differing properties of the detection platforms and the physiological state of the circulating anti-NDO-LID antibodies in each patient group.

Alongside regular monitoring of the serological response, researchers also conducted follow up skin slit smears to determine impact of MDT on BI. BI were markedly higher in patients that would experience ENL than the other groups, whereas patients that did not experience reactions had similar BI to patients that developed RR at the time of diagnosis. MDT prompted decline in BI for most patients such that it was significantly lower after 12 months after the initiation of MDT that the levels observed at time of diagnosis. Although significantly reduced BI were, however, still positive in most cases at the end of MDT. The declines continued beyond the actual MDT administration phase and at 2, 3 and 4 years of assessment BI were significantly lower in patients that experienced RR than those measured in patients that had not reactions. Also of note, relative to the other groups, patients that experienced ENL had significantly higher BI at each assessment time and the average BI was still >1 even 4 years after MDT initiation.

In addition to monitoring patients,  a cohort of contacts was also monitored by clinical and serological examinations in order to detect emerging patients. 7 clinically confirmed patients emerged from the contact surveillance group. Two of these contacts developed TT leprosy (BI = 0) and were accordingly seronegative. The other 5 developed BT leprosy, with 3 of the 5 having positive results in RDT at numerous points in their monitoring schedules. This composition (5 BT; 2 TT) was a markedly different patient presentation than observed among those 200 individuals enrolled into the project on the basis of clinic attendance and confirmation as leprosy patients (88 LL; 78 BL; 0 BB; 34 BT; 0 TT). Although only 3 of these emergent patients were positive by RDT, the data indicate that being test positive indicates elevated (but not absolute) risk of developing leprosy relative to the RDT negative contact and general populations.

Impact

Comparative evaluation of antibody detection tests to facilitate the diagnosis of multibacillary leprosy. Duthie M, Orcullo FM, Abbelana J, et al. Applied microbiology and biotechnology. 2016;

Need for, and acceptability of, rapid diagnostic tests that can facilitate the diagnosis of leprosy. Duthie M, Orcullo FM, Maghanoy AA, et al. Leprosy review. 2016; 87 (2) : 158-70.

Combination chemoprophylaxis and immunoprophylaxis in reducing the incidence of leprosy. Duthie M, Balagon M. Risk management and healthcare policy. 2016;

Utility and limitations of serodiagnostic tests in monitoring the response to treatment of leprosy patients. Duthie M, Roferos F, Abellana J, et al. Diagnostic microbiology and infectious disease. 2020;

Presentations at International Leprosy Congress, Manila, September 2019:
- Sustained acceptability of use of rapid diagnostic tests among patients and their contacts in the Philippines. Malcolm S. Duthie, Florenda O. Roferos, Armi Maghanoy, Marivic F. Balagon
- Utility and limitations of serodiagnostic tests in monitoring the response to treatment of leprosy patients. Malcolm S. Duthie, Florenda O. Roferos, Junie F. Abellana, Tonet Taborada, Armi Maghanoy, Marivic F. Balagon