These rapid diagnostic assays detect the presence of leprosy bacilli proteins or host antibodies against them. If adequate quantities of the target antigen are present in the sample, it will bind to specific antibodies attached to a paper strip which will produce a visually detectable signal within 30 minutes. The observed antigen(s)/antibodies, which can only be expressed if bacteria are present in the host and have a high BI index, are thus usually used for acute or early infection detection (14, 15). Antibody detection is a more general form of leprosy diagnosis test, which measures the presence of antibodies in the blood of people suspected to be afflicted with Mycobacterium leprae. Specific antibodies are made within days or weeks after initial exposure. The intensity of the antibody response relies on a variety of factors such as bacillary load, type of immune response as well as other host factors such as age, nutritional status, disease severity and certain medications or infections such as HIV suppresses the immune response (16). Though antibody levels provide some insight into the status of infection, but they are insufficient to distinguish between recent and past infections. However, finding the evidence of infection would suggest a recent transmission (17).

M. leprae can be specifically detected by PCR or qPCR techniques in a very sensitive manner i.e. for detecting a very small number of genomic templates in a clinical sample. Several specific genomic regions have been targeted such as 16S rRNA (7) rpoT and sodA (18, 19) 36-kDa antigen (20), Complex 85 (21) and the repetitive sequences (LEPRPT, LEPREP, REPLEP and RLEP) (22) among other M. leprae genes (23–25) of which the most sensitive and specific sequence was found to be RLEP region (26). Other repeated loci can also be targeted that are present in fewer copies. Leprosy is often diagnosed by molecular testing either from PCR or qPCR (7, 8, 27). NAAT evaluations are focused on amplifying the M. leprae’s targeted sequence of the genetic material (DNA or RNA) with PCR. NAATs are more rapid than conventional mycobacterial detection technologies and are also available at different healthcare levels, therefore allowing them to perform drug susceptibility testing, such as rifampicin (RIF), for the detection of leprosy (28). In this way, they simplify leprosy diagnostics and help improve the quality of leprosy treatment.

The immunological spectrum of leprosy, which ranges from widespread infection to a self-limited type of disease, often complicates the diagnostic procedure. The intricate interaction between innate and adaptive immune responses, which is controlled by host genetic background and environmental variables, determines M. leprae infection and subsequent disease development. Biomarkers based on the host immune response to M. leprae are thus a topic of interest as detecting M. leprae in the preclinical phases of the disease is challenging. Many research groups have recognized the potential of M. leprae specific antigens in serologic and T cell tests since the disclosure of the M. leprae genome. Despite the screening process to identify the early candidate that triggers M. leprae specific T lymphocytes, suitable candidate antigens are yet to be identified that would cover the entire immunological leprosy spectrum.

In this review, we have tried to compile all the information available in the literature regarding the recent developments for leprosy diagnosis (Table 1). We refer readers to other reviews to know about the recent advancements in biomarker-based research for leprosy detection (65–68) and meta-analysis for sensitivity and specificity studies of laboratory tests (10, 29–31).

Following the publication of the genome sequence of the M. leprae in 2001, species-specific genetic sequences have been sought to standardize the diagnostic procedures based on DNA analysis. These sequences were amplified using the PCR method, allowing the identification of bacillus DNA from modest numbers of M. leprae cells. PCR and quantitative PCR (qPCR) are extremely informative and responsive and guarantee to be diagnosed and treated early enough to guarantee the timely medication required to avoid disability and to mitigate leprosy spread (7, 71). The PCR-based approaches used to detect pathogenic DNA and RNA can also assess the viability of leprosy bacilli and be used in touch screening and monitoring programs. Several tissue origins, including skin biopsy tests, swabs of the nasal or dental, and entire blood, can be extended with PCR for M. leprae DNA. However, the use of skin biopsies instead of conveniently obtained samples (blood, skin scrapings, saliva etc) provides maximal performance (24).

Although M. leprae was considered the exclusive causative agent of leprosy, recently, M. lepromatosis was discovered and found in an unusual type of leprosy known as diffuse lepromatous leprosy (DLL) (94). A unique repetitive factor, RLPM, was defined by genomic analysis of M. lepromatosis strain (NHDP-385) on which a real-time quantitative PCR (qPCR) assay was established and validated in compliance with the guidelines for Clinical Laboratory Improvement Amendments as clinical diagnostic assays (27). Similarly, a new assay was developed that can simultaneously detect both the leprosy bacilli through PCR in a single reaction by amplifying a part of rpoT gene (19). Therefore, in clinical diagnosis and monitoring of leprosy in the field settings, the RLPM (M. leprae), RLEP (M. lepromatosis) and rpoT based PCR, 16SrRNA, sodA, RLEP based PCR (18) and qPCR assays can support healthcare providers. The availability of a rapid, specific and sensitive, and field-deployable PCR assay would support local decision-making during the surveillance and screening period (95). Few PCR examples are already reported such as Portable PCR (58), Palm PCR (57), insulated isothermal PCR (iiPCR) (60), RT-insulated isothermal PCR (96), Backpack PCR (59), etc. These assays have been successfully used for several pathogens detection and can also be adapted for the detection of leprosy bacilli.

Endemic areas of leprosy are often lacking specialized labs that highlight the need for low-complexity diagnostics (97–99). A test was developed and field-assessed known as the lateral flow test (100). Recently, Upconverting phosphor (UCP) reporter technology was used in all LFAs to increase sensitivity and quantitation (101). The test consists of the UCP technology in conjunction with low-cost immune chromatography (i.e. lateral flow). It performs exceptionally well for the identification of cytokines and anti-M. leprae PGL-I IgM Ab (αPGL-I) in serum. A quick test using the visual detection of immunogold particles has been used to detect antibodies against M. leprae (93). No sophisticated analytical laboratory equipment is required for the user-friendly UCP-LFAs. This low-cost, lightweight portable reader offers complete, instrument-assisted analysis and prevents operator interference. Using UCP-LFA, the quantity of every biomarker type present in biological samples may also be quantified, which will enable the measurement of variations in the quantities of biomarkers and adjustable cut-off values to fulfill exposure and specificity criteria for areas where the leprosy is endemic (92, 102).

Isothermal amplification is a simple process that rapidly amplifies nucleic acid sequences at constant temperature (103). It is favored over typical PCR approaches to get rid of the specifications/complications of a costly thermocycler system and is cost-effective. The loop-mediated isothermal amplification (LAMP) technique has many applications in the area of point-of-care (POC) research across many isothermal techniques (104). It uses four to six primers to determine particular temperature regions which can be multiplied at a constant temperature using high strand displacement DNA polymerase. Gene amplification and detection can be achieved in one step, using the basic heat block or water bath, by the incubation of the polymerase primer and template mix in a reaction buffer. It can obtain outcomes in less than 30 minutes at a single temperature with a high yield and specificity. LAMP is considered to be useful for many molecular diagnostic applications i.e. from laboratory infectious agents to different pathogens, food processing, environmental examinations etc. (105, 106). Several LAMP-based diagnostic kits and approaches have been developed to identify a variety of challenging pathogenic agents for highly infectious illnesses, including SARS-CoV-2 (107). More importantly, the advent of LAMP has led to improved diagnosis of neglected tropical diseases all over the world. Esmatabadi et Al checked different methods for identification that can be paired with the LAMP system to interpret results quickly, such as colorimetry, turbidometry, hybridization samples, lateral flow dipsticks, ELISA, gold nano-particles (108). M. leprae is uncultivated in vitro and has a highly reduced genome that has a high degree of identification with the MTB genome. Considering its unique advantages that provide a rapid and low-cost diagnostic tool for disease detection in poor and remote parts of the world, recently LAMP technology for M. leprae detection has been developed using RLEP sequence as a molecular target for this assay (61–63). However, no isothermal amplification assay has been developed for M. lepromatosis yet.

The NDO-LID® test, a new ELISA-based diagnostic test, was developed for the diagnosis of MB leprosy. The test is based on the detection of antibodies against the unique protein-glycolipid complex. The NDO-LID® immunochromatographic test takes only a tiny sample of serum or whole blood. This test includes both the LID-1 and PGL-I antigens. These antigens are immobilised on nitrocellulose membranes, which allow for the transfer and detection of the specific antibodies against these antigens in patient sera. This test, when combined with a new mobile based platform (Smart Reader® application), can give measurable and reliable data to aid in leprosy diagnosis. It detects MB patients and, perhaps, HHC at a higher risk of acquiring MB leprosy, much as the other leprosy serological tests (32, 109–112).

It is the first PCR-based diagnostic test for leprosy that follows Good Manufacturing Practice (GMP). This multiplex real-time PCR test has been developed to detect M. leprae 16S rRNA and RLEP genes, as well as one mammalian target (18S rRNA gene) that acts as a control. This detection occurs by the increase, at each reaction cycle, of the fluorescence signal emitted by two molecular probes specific when the target DNA is present in the sample. The success of the reaction is monitored through a fluorescence signal, emitted by a third probe in the same reaction, which increases the amplification of DNA. The Brazilian Health Regulatory Agency (Anvisa) approved the NAT-HANS test for people suspected of leprosy in 2021. The test showed high sensitivity and specificity values comparable to Lateral Flow Assays with a limit of detection of 2.29 copies of the M. leprae genome (33, 34).

Dapsone is an anti-inflammatory and antibacterial medication used in dermatology to prevent and treat infectious and chronic inflammatory conditions (113). Dapsone is used to treat leprosy, malaria, and diseases as an antibiotic and as an anti-inflammatory medication but is most commonly utilised for its anti-leprotic action. However, there have been reports of Dapsone Hypersensitivity Syndrome (DHS) which poses serious concern in certain populations. HLA-B*13:01 is associated with DHS and prior screening is essential to significantly minimize the chances of occurrence of DHS. A single DNA polymorphism termed HLA-B*13:01 located in the human leukocyte antigen locus is more common in those who developed DHS (35–37). Individuals with one copy of HLA-B*13:01 risk variant are 34 times more likely to develop DHS than individuals without it. Two copies of HLA-B*13:01 make individuals times more susceptible to the syndrome (75). Using TBG Biotechnology Corp’s HLA-B*13:01 assay kit, Clinicians and researchers are finally able to screen the patients for DHS. Recently, HLA-B*13:01 is validated as a biomarker for DHS in leprosy patients and Screening for HLA-B*13:01 has shown promising results in reducing DHS incidence significantly in the Indonesian population (114). The kit also provides the typing results of HLA-A, B, C, DRB1, DRB3, DRB4, DRB5, DQB1 and DPB1 with high resolution by a DNA-based method (http://www.tbgbio.com/en/product/product_detail/12).

A test was developed in 2012 as a GenoType LepraeDR kit using DNA strip technology (detection of amplified DNA or RNA sample via hybridization and alkaline phosphatase reaction on a membrane strip) to overcome this and extended to the molecular diagnosis of antibiotic resistance in leprosy (https://www.hain-lifescience.de/en/products/microbiology/mycobacteria/lepra/genotype-lepraedr.html). The GenoType LepraeDR enables the simultaneous identification of M. leprae and its susceptibility to first and second-line drugs within 5 hours. Initially, 120 M. leprae strains analyzed for genotypic and phenotypic characters of resistance were routinely screened for evaluation. The assay findings were found to be 100% compatible with those of the in vivo susceptibility test, while the PCR sequencing results were 98.3% for rifampicin, 100% for Dapsone, and 100% for fluoroquinolones (28). Later many publications have described this assay to identify the drug resistance in M. leprae (29, 38–40).

The Primerdesign genesig Kit^® for Leprosy is intended for quantification of M. leprae genomes in vitro. The kit is intended to provide a wide detection profile. Their primers show 100 percent homology with more than 95 percent of the NCBI database reference sequences available at the time of creation. Because of the dynamic nature of genetic diversity, additional sequence information may become accessible after the initial design. The method employs a TaqMan probe to perform real-time PCR of the non-coding repetitive element RLEP, which is unique to the M. leprae genome. The excellent test specificity was demonstrated utilizing pathogenic and opportunistic pathogenic mycobacterium reference DNA samples, as well as PCR detection of single-copy genes of M. leprae such as fbp, MntH and rrs. The application of the developed approach increased the sensitivity of a commercially available test system based on single-copy rpoB gene detection to 96.8%, while the use of a commercially available test system based on single-copy rpoB gene detection provided 59.4 percent sensitivity to the detection of M. leprae in clinical material (41).

American Leprosy Mission and its network of research partners have collaborated with Biomeme, Inc. to develop and commercialize hand-held point-of-need PCR testing solutions for combatting leprosy diagnostics for use in distant locations. Their FranklinTM mobile qPCR thermocycler and field-prepared, shelf-stable reagents are easy-to-use reagents for field-friendly operations (https://biomeme.com/). Biomeme uses a polymerase chain reaction (PCR) methodology that turns a smartphone into a powerful mobile lab capable of performing PCR, RT-PCR, qPCR, and isothermal assays in real-time. A Biomeme mobile qPCR thermocycler, which is smaller than a box of tissues, is at the core of the solution. The lightweight, internally battery-powered gadget produces results in 30-45 minutes without the need for electricity, centrifugation, or cold chain logistics (https://www.ccih.org/technology-in-global-health-an-instrument-of-impact/). The use of the Biomeme platform as a field-friendly PCR-based early diagnostic tool to detect subclinical infection among household contacts and the general population has been rolled out in some parts of the world (India, Nepal and Ghana).

Leprosy in endemic regions has been diagnosed based on the presence of one of 3 cardinal signs: skin lesion with loss of sensation associated or not with thickened peripheral nerves and microscopic detection of acid-fast bacilli in skin lesions/slit skin smears. Keeping in mind, WHO released a manual on neglected tropical diseases by skin changes in 2018 (115). This serves as a training guide for the front-line health professionals, a detailed guide to recognizing skin NTDs employing signs and skin symptoms offering guidance about how to detect and cope with serious skin conditions that are faced by unspecialized front-line health care personnel. This pictorial training guide on neglected tropical diseases of the skin (including leprosy) has been recently converted into an interactive mobile phone application. The program allows easy access to the knowledge about skin conditions – such as its clinical symptoms, management and geographical distribution – which provides a list of possible indications to health care professionals and the general public. The software also facilitates rapid knowledge exchanging through a chatbox, which will promptly answer general questions. In collaboration with national dermatological federations, the SkinApp has been evaluated in field settings in Ethiopia, Mozambique and Tanzania under the PEP4LEP initiative in 2019. During their daily clinical activity, the primary health staff found it very helpful (42, 43).

LEARNS is a mobile-based leprosy identification system that enables primary health providers to send an SMS to a specialist with photos of suspected leprosy and symptoms. This helps in a dramatic reduction in the time required for testing and treatment times. Beginning in the Philippines, where nearly 2,000 new leprosy patients have been identified in leprosy annually, LEARNS was initiated with the support of a Leprosy Task Force as part of the Philippines Department of Health (DOH)-Novartis Foundation. In 2015, A diagnostic compatibility between LEARNS and the clinician-guided diagnosis was determined. LEARNS has proven to be a successful screening method for the proper detection of suspected lesions as 83 percent leprosy and 77 percent leprosy except for specifics, where the suspected lesions have been presented in the picture. About 3,500 healthcare providers have been qualified nationally in LEARNS to date (https://www.novartisfoundation.org/past-programs/accelerating-leprosy-and-malaria-elimination/leprosy-alert-and-response-network-system-learns). In line with LEARNS, similar computer-based software application like NIKUSHTHA which is developed to report leprosy in India has been started working. (https://dghs.gov.in/content/1349_3_ NationalLeprosyEradicationProgramme.aspx) whereas a Portuguese mobile application system is in development for detecting leprosy suspects (116).

Artificial intelligence (AI) has become a hot topic among doctors in recent years with the evolution of information technology and interdisciplinary studies have become more common. Accurate identification of leprosy lesions is often a challenge, especially for clinicians with little experience such as those in low-burden settings. To address this problem, the convolutional neural networks (CNN) project was developed using DermnetNz datasets in 2016, achieving 91.6 percent accuracy (117). In 2018, researchers used the Kohonen Self-Organizing Maps algorithm to evaluate data from patients and their household connections using Artificial Intelligence approaches to study the epidemiology of leprosy (118). The results show a significant frequency of late diagnosis, as well as Anti PGL-1 levels in clusters, indicating a higher bacillary load and consequently a high risk of disease progression. Microsoft and Novartis Foundation have also teamed up to develop an AI-powered digital tool for the early detection of leprosy (https://www.novartisfoundation.org/news/ai-powered-diagnostic-tool-aid-early-detection-leprosy). They have named it AI4Leprosy project which aims to develop an easy-to-use AI-powered solution to accelerate the diagnosis of leprosy with skin images. Recently, a cross-platform app for leprosy screening based on artificial intelligence has been developed which can recognize patterns of leprosy cases in a database with ~94% sensitivity and 87% specificity (45). Recently, Barbieri et al. described that AI models have the potential to become a complementary diagnosis tool, especially for the remote areas where very few clinicians are available known as AI4Leprosy (46). It is an AI-enabled image-based diagnosis tool for leprosy that is based on a mix of skin images and clinical data collected using a defined method. For leprosy diagnosis, it had a high classification accuracy (90%) and an area under curve (AUC) of 96.46 percent.