LGCmain
Indian Journal of Pathology and Microbiology
Home About us Instructions Submission Subscribe Advertise Contact e-Alerts Ahead Of Print Login 
Users Online: 1261
Print this page  Email this page Bookmark this page Small font sizeDefault font sizeIncrease font size


 
  Table of Contents    
REVIEW ARTICLE  
Year : 2022  |  Volume : 65  |  Issue : 5  |  Page : 300-309
Laboratory perspectives for Leprosy: Diagnostic, prognostic and predictive tools


Department of Pathology, Dr. Ram Manohar Lohia Institute of Medical Sciences, Gomti Nagar, Lucknow, Uttar Pradesh, India

Click here for correspondence address and email

Date of Submission06-Nov-2021
Date of Acceptance23-Dec-2021
Date of Web Publication11-May-2022
 

   Abstract 


The diagnosis of leprosy poses several challenges. The bacillary load, serology, and tissue response are determined by the host immune status, which make individual tests unsuitable across the spectrum. The sensitivity of tests for identifying paucibacillary cases remains limited, on the other hand, many tests lack specificity in differentiating contacts from diseased cases. Nonetheless, a plethora of laboratory tests have been added to the armamentarium of the clinicians dealing with leprosy. In the current review, we critically analyze the tests available for diagnosis, prognostication, and prediction of treatment response in leprosy. We discuss in brief the conventional tests available and detail the newer serologic and molecular tests added over the past few years with an attempt to suggest the pros and cons of each, and the tests best fit for each clinical scenario. Slit skin smears and skin or nerve biopsies are primarily performed to exclude clinical mimics, confirm a diagnosis, and immunologically subtype the case. Antibody titres of phenolic glycolipid-1 and its synthetic variants can be measured in serum and saliva and provide noninvasive means to detect leprosy with good specificity. Conventional, quantitative, real-time, and other variants of PCR can detect M. leprae DNA and have been used to effect in blood, tissue, and urine samples. T helper I and II cytokine signatures can be used to differentiate the subtypes of leprosy. Newer machine learning algorithms use combinations of these tests to predict the development of leprosy in contacts. Tests to detect treatment response, antimicrobial drug resistance, and predict the onset of reactions in leprosy can be used to advantage. We compare the characteristics of these tests and suggest an algorithm for leprosy diagnosis optimally utilizing them in various clinical settings.

Keywords: Cytokines, diagnosis, drug resistance tests, leprosy, polymerase chain reaction, prognosis, serologic tests

How to cite this article:
Malhotra KP, Husain N. Laboratory perspectives for Leprosy: Diagnostic, prognostic and predictive tools. Indian J Pathol Microbiol 2022;65, Suppl S1:300-9

How to cite this URL:
Malhotra KP, Husain N. Laboratory perspectives for Leprosy: Diagnostic, prognostic and predictive tools. Indian J Pathol Microbiol [serial online] 2022 [cited 2022 May 24];65, Suppl S1:300-9. Available from: https://www.ijpmonline.org/text.asp?2022/65/5/300/345040





   Introduction Top


The scourge of leprosy has existed from time immemorial. It is a devastating disease caused by Mycobacterium leprae which attacks the immune system with manifestations in the peripheral nerves, skin, joints, and internal organs. The World Health Organization statistics place India among the worst-hit countries accounting for 114,451 of the 2,02,185 new leprosy cases detected worldwide in 2019. Remarkably, the cases detected with grade 2 disability remain high. The need of the hour is to control transmission through early and correct detection and prompt adequate treatment.[1]

Challenges in leprosy diagnostics are numerous. One major impediment for fruitful leprosy research has been the inability to culture the M. leprae bacillus in vitro. There is a noteworthy difficulty in differentiating household contacts from diseased patients.[2] The sensitivity of tests for identifying paucibacillary cases remains limited.[2] Also, the immune spectrum of leprosy offers challenges in presenting an array of serum markers and variable bacillary load which makes individual tests unsuitable across the spectrum. Nonetheless, a plethora of laboratory tests have been added to the armamentarium of the clinicians dealing with leprosy.

In the current review, we have attempted to exhaustively analyze the newer tests available for all three significant aspects of leprosy – its diagnosis, prognostication, and prediction of treatment response. Early detection of lepra reactions and determination of drug resistance are the new challenges pathologists now encounter. We discuss in brief the conventional tests available and detail the newer molecular tests added over the past few years with an attempt to suggest the pros and cons of each and the tests best fit for each clinical scenario. [Table 1]
Table 1: Key messages pertaining to laboratory diagnosis of leprosy

Click here to view


Diagnostic tests

An ideal diagnostic test for leprosy would be a noninvasive test, applicable for use by field workers, sensitive, specific for leprosy, and able to differentiate household contacts from cases. Although no single optimum test exists, many tests have attempted to fulfill these criteria. Conventional tests in leprosy have focused on identifying the bacillus. These include slit skin smears and skin or nerve biopsies. These are primarily performed to exclude clinical mimics, confirm a diagnosis, and immunologically subtype the case. The lepromin test detects the immune response of the host to infection by lepra bacilli. Other tests having potential diagnostic use include those that identify the M. leprae bacillus or its genetic sequence (e.g. molecular tests) and those that identify the antibodies induced by the bacillus.

Slit skin smear (SSS)

This test is considered the gold standard for leprosy diagnosis. It is recommended for confirming a diagnosis in cases with clinical suspicion of leprosy or in diagnosed cases that relapse after treatment completion. It involves collecting smears from various lesional and nonlesional sites, identifying lepra bacilli, and determining a bacteriological index (BI).[3] The BI is a logarithmic indicator of the density of bacilli in the smears.[4] It corresponds to the immunologic state of the patients, and the subtype of leprosy.[4] The SSS is an inexpensive test that can be used by field workers with limited expertise. Though SSS is considered a 100% specific test for leprosy diagnosis, its sensitivity is limited and ranges from 10% to 50%, depending on the expertise of the operator.[3] A study in Brazil found SSS positivity in 35.9% of newly diagnosed leprosy cases only.[5] Although sensitivity was 59.8% in multibacillary cases, paucibacillary cases showed a significantly lower sensitivity on SSS (1.8%) as compared to PCR (75.4%) performed on skin biopsy material in a study by Banerjee et al.[6]

Skin biopsy

Skin biopsies from clinically suspicious lesions enhance the information that can be gathered from a SSS. They allow assessment of the histologic subtypes – polar tuberculoid (TT), borderline tuberculoid (BT), mid borderline (BB), borderline lepromatous (BL), and polar lepromatous (LL). [Figure 1] The histologic features of various subtypes of leprosy have been discussed comprehensively by Massone et al.[7] The clinical subtypes and those assessed on histology show agreement in 58% to 70% of the cases; the disagreement is greater in the borderline subtypes.[7],[8] Though multibacillary cases can be definitively diagnosed by identifying bacilli, granulomatous histology on biopsy can support a clinical suspicion of paucibacillary leprosy. Various clinical mimics of leprosy can be excluded by the use of skin biopsies. These include pityriasis alba, tuberculosis and postinflammatory hypopigmentation which mimic TT; sarcoidosis and granuloma annulare which mimic BT; morphea, parapsoriasis, and necrobiosis lipoidica which mimic BB and BL; juvenile xanthogranulomatosis, and chromoblastomycosis etc., which mimic LL.[9] Biopsies also help ascertain the presence or absence of reactions. The presence of Type I reactions is suggested by edematous coalescing granulomas and that of Type II reactions by vasculitis or subcutaneous infiltrate.[7] Rare atypical presentations of leprosy can be definitively diagnosed by the use of skin biopsies.[10],[11] Histoid leprosy which presents clinically as disfiguring nodules on a backdrop of unremarkable skin, shows intense infiltrates of spindled, vacuolated, or epithelioid cells with very high bacillary indices.[10] Lucio phenomenon which presents as irregular nonblanchable extremity purpurae or ulcers, shows ulcerated epidermis, vessel wall infiltrates of lepra bacilli, and vessel wall thrombosis.[11]
Figure 1: (a) Section from peripheral nerve with TT leprosy showing two nerve funicles with central necrosis (asterisk) surrounded by epithelioid cell granulomas rimmed by lymphocytic collars. (b) Skin biopsy with BT leprosy showing a collection of epithelioid cells (arrow) with admixed lymphocytes. (c) Skin biopsy with BB leprosy with diffuse infiltrate of lymphocytes, epithelioid cells (line), foamy histiocytes (arrow), and giant cells (asterisk). (d) Skin biopsy with BL leprosy showing foamy histiocytes (arrows) admixed with lymphocytes surrounding dermal neurovascular bundles. (e) Skin biopsy with LL leprosy showing foamy histiocytes (arrows) surrounding dermal neurovascular bundles. (f) Numerous lepra bacilli and few globi (arrows) in the dermis from a case of LL leprosy (a-e) Hematoxylin and Eosin, ×100; (f) Wade Fite, ×1000)

Click here to view


Nerve biopsy

Nerve biopsies though relatively invasive out-patient procedures are valuable tools for diagnosing pure neuritic leprosy (PNL). A case of PNL is suspected based on clinical features, nerve thickening, electroneuromyography (ENMG), and High-resolution ultrasonography (HRUSG) studies. The presence of bacilli in nerve biopsies constitutes definite evidence of leprosy. However, in the absence of bacilli as is a common occurrence, in paucibacillary cases or where treatment has been initiated before biopsy, nerve biopsies can still be useful adjuncts in diagnosis. These help in excluding clinical mimics of vasculitic neuropathy, chronic inflammatory demyelinating neuropathy, amyloidosis, etc., that may variably present as mononeuropathy multiplex.[12],[13] Lepra bacilli have been identified in 24.3% to 47.8% of leprous nerve biopsies in various reports.[14],[15],[16],[17] In a south Indian study, 46 cases of PNL were biopsied and more than half of these (52.17%) did not reveal lepra bacilli on histology. However, they suggested the presence of inflammatory infiltrate in the nerve bundles, fibrosis, thickening of perineurium, and myelinated fiber loss as features suggestive of leprous neuropathy in absence of bacilli in nerve biopsies.[14] [Figure 1]

High resolution Ultrasonography (HRUSG) and Magnetic Resonance Imaging (MRI) studies

Though a detailed review of radiology as a diagnostic modality in leprosy is outside the scope of this review, mention must be made of their increasing contribution in leprosy diagnostics. HRUS and MRI are of value in peripheral nerve studies, especially in PNL cases. High-frequency linear array transducers are used to read the echo density of peripheral nerves. These can assess enlargement of the nerve or its fascicles, epineurial thickness, and endoneurial vascularity in the nerves.[18] Shukla et al.[19] in a study on 100 PNL cases, found HRUSG to be more sensitive than clinical examination for the determination of nerve thickening. They also found the polar LL cases to show significant hypoechogenicity and vascularity on HRUSG. Increased nerve vascularity on Doppler studies or increased intensity on T2 MRI and Gadolinium enhancement have been shown to be associated with lepra reactions.[20]

Lepromin test

The Mitsuda reaction is used to assess the histologic cell-mediated immune response of an individual to an intradermal injection of M. leprae antigens. The test shows a fair correlation with the clinical subtype, being positive in paucibacillary and negative in multibacillary cases. However, positive results have been reported in up to 33% of BL and LL cases. The lepromin test correlates negatively with the level of serum phenolic glycolipid (PGL)-1 antibodies.[21] The relative risk of developing leprosy was eight times higher amongst household contacts with negative Mitsuda reaction, absence of scars after BCG vaccination, and presence of serum antibodies to PGL-1 antibodies.[22] Though the test is of historic importance for leprosy diagnosis, lepromin-negative candidates in a population may be offered prophylactic vaccines for leprosy.[23]

Immunological tests: Humoral immune response

Several antigenic determinants of M. leprae induce a humoral response in the host. These include native and synthetic antigens like PGL-1, Lipoarabinomannan, Major membrane protein-II, Infectious Disease Research Institute Diagnostic-1 (LID-1), natural disaccharide antigen octyl (ND-O), and others.[24],[25] PGL-1 is a component of the M. leprae cell envelop consisting of a trisaccharide specific to M. leprae.[24] It has been most extensively evaluated as a diagnostic serum marker in leprosy. The ND-O is an artificial substitute for PGL-1 and has also been studied as a serologic marker.

Types of tests used

Serum antibodies produced against these antigens have been quantified using several tests, each with different specificity and sensitivity. A metanalysis by Gurung et al.[26] identified 78 studies of which 39 estimated PGL-1 IgM antibodies using enzyme-linked immunosorbent assay (ELISA) with a summary sensitivity of 63.8% and specificity of 91%. The ML flow test which uses lateral flow immunochromatography was analyzed in nine publications and showed summary sensitivity of 67.9% and specificity of 86.7%. The agglutination test showed better summary sensitivity (72.8%) and specificity (90.1%).

Types of antigens tested

The PGL-1 ELISA is a useful and affordable quantitative test.[27] However, natural PGL-1 is hydrophobic and simpler tests have been developed utilizing its synthetic variants.[28] A simple dipstick method utilizes ND-O conjugated with bovine serum albumin for antibody detection.[29] The particle agglutination assay on the other hand uses gelatin particles coated with synthetic trisaccharide moiety of PGL-1.[28] The NDO- leprosy infectious disease research institute (IDRI) diagnostic (LID) test combines the synthetic ND-O and LID-1 antigens, and has been used in rapid leprosy diagnostic tests.[27],[30] Some of these kits can be evaluated by an android smartphone–based reader and are rapid and easy to perform.[30] The sensitivity in diagnosing leprosy was reported to range from 87% in multibacillary to 32.3% in paucibacillary cases, with a reported specificity of 97.4%.[30] Results of a metanalysis however did not find any significant difference in sensitivity or specificity of tests using different antigens.[26]

Few newer antigens are currently under research. These include early secretory antigen target (ESAT-6), ML0049, and CFP-10 (lepra culture filtrate protein-10 (CFP-10) which induce an interferon response in the host. These show homology with M tuberculosis and cannot distinguish the two in endemic regions.[31]

Utility of PGL-1 serology

Numerous studies have found a good correlation between PGL-1 ELISA and the bacteriological index. A systematic review of PGL-1 has shown that it is of value in classifying leprosy subtypes.[32] The common shortcoming of all serologic tests is their lack of sensitivity for detecting individuals with early or paucibacillary leprosy.[31] Higher specific IgM antibody titers develop in cases with BL and LL forms as compared to TT cases. An average of 78% multibacillary and 23% paucibacillary leprosy cases show positive PGL-I antibodies in serum.[23],[31],[32] Given the low positivity in paucibacillary cases, PGL-1 serology may not be a measure of infection prevalence.[33] Up to 18.4% of leprosy patients contacts test positive with serologic tests, indicating subclinical infection.[32] This may be of benefit in identifying infected contacts for leprosy control programs as it has been noted that seropositive contacts have a 7.2 fold increased risk of developing symptomatic leprosy.[34]

In a study on pure neural leprosy, lower PGL-1 ELISA titers significantly correlated with involvement of a larger number of nerves with higher sensory and motor symptoms, indicative of greater cellular rather than humoral immune responses in PNL. Polymerase chain reaction (PCR) values in these cases were also lower.[35]

Molecular tests: Polymerase chain reaction

Over the past few years, PCR has evolved as a detection method for M. leprae DNA, akin to its constructive use in the diagnosis and monitoring of tuberculosis. Though expensive and not readily available at the field levels, it is a versatile tool for leprosy. It is useful in clinically suspicious or atypical cases where skin smears and/or biopsies are noncontributory to the diagnosis.[36] Studies comparing PCR to microscopic detection of lepra bacilli have suggested improved detection using PCR (mean PCR positivity 44% versus 30% for microscopy). A synthesis of over 26 studies on multibacillary and 28 studies on paucibacillary cases reveals that the mean detection rate is 77% for PCR versus 59% for microscopy, and 48% for PCR versus 23% for microscopy, respectively.[37] Azevedo et al.[38] found positive PCR results in 55% of polar tuberculoid and 95% of borderline tuberculoid cases, suggesting helpful use of PCR in this subset which is normally smear negative. In a recent metanalysis, PCR showed the best sensitivity among different techniques used to detect paucibacillary leprosy.[39] PCR shows some increment in accuracy when compared to serum ELISA.[26]

Types of tests used

Conventional PCR has been used in most studies evaluating the utility of PCR in leprosy diagnosis. Multiplex PCR, quantitative real time PCR (qPCR), nested PCR, and reverse transcriptase PCR (RT-PCR) have all been fruitfully used.[37] The 18kDa, 36kDa, 65kDa, M leprae-specific repetitive element (RLEP) DNA sequences and 16Sr RNA are specific for M.leprae and have been tested.[23],[36] In a metanalysis, conventional PCR used in seventeen studies showed summary sensitivity and specificity of 75.3% and 94.5%, respectively. Quantitative PCR was evaluated in five studies and showed better sensitivity (78.5%) but lower specificity (89.3%) than conventional PCR. Studies using RLEP for conventional PCR showed similar sensitivity but better specificity implying optimism for developing RLEP as a diagnostic marker for leprosy. [26],[37] Recent reports of droplet digital PCR show enhanced sensitivity over qPCR in diagnosing paucibacillary cases.[40]

Types of samples tested

Leprosy PCR has been performed on various clinical samples. These include skin, nasal mucosal, and nerve biopsies, nasal swabs and slit skin smears, blood and urine samples.[41],[42],[43],[44],[45],[46],[47] Mean PCR positivity was 61% when performed in SSS versus 36% for microscopy and 70% when performed in skin biopsies versus 44% for microscopy.[37] Santos et al.[35] studied pure neuritic leprosy cases and found thickened nerves in 58.6%, anti PGL-1 ELISA in 52.9%, and positive qPCR in 60.8% of cases. In a study by Tiwari et al.,[42] 37.14% of PNL cases showed lepra bacilli on nerve biopsy, whereas 62.8% were positive on PCR. No significant difference in specificity or sensitivity of leprosy diagnosis was seen between different samples or methods of DNA extraction. The bacillary load (which is expected to be similar for all body sites in multibacillary patients) is the primary determinant of the sensitivity of the test.[26] PCR has been effectively used both in fresh tissue biopsies and also in tissue retrieved from formalin-fixed and paraffin-embedded tissue blocks. The latter technique can be a good salvage measure where a differential of clinically unsuspected leprosy is considered on histopathology or the biopsy does not reveal bacilli. [48],[49]

Cytokine and chemokine profiles

Cytokine profiles in leprosy have been a matter of research in the past few decades. Some of these show significant differences among various subtypes of leprosy or among different reactive states. However, their current diagnostic utility is not well established. Geluk et al.[50] found considerable differences in IL-1b, MCP-1, and MIP-1b levels in asymptomatic contacts versus diseased leprosy patients. Higher levels of interleukin-1beta (IL-1b) and monocyte chemoattractant protein-1 (MCP-1) have been reported in TT as compared to LL cases. T-helper 1 cytokine expression (IFN gamma, IL2) has been found in RNA extracts from skin lesions of paucibacillary leprosy and T-helper 2 cytokines (IL4, IL5, IL10) in multibacillary leprosy.[51] PB cases have also shown higher serum levels of IFN-gamma and TNF-alpha as measured by ELISA, whereas MB cases show significantly higher IL-1 beta and IL-10 levels.[52] Such biomarker signatures may be of use in differentiating subtypes of leprosy. Multiplex lateral flow assay formats utilizing several such biomarkers together are currently under research.[31] Tuberculoid leprosy patients show an IFN gamma response to early secretory antigenic target (ESAT)-6 and culture filtrate protein-10 (CFP-10) M. leprae antigens. However, tuberculosis cases and healthy controls from endemic areas also test positive, limiting its utility as a diagnostic test.[31]

Risk assessment in contacts

Tests used to diagnose leprosy often show positivity in household contacts of diseased cases. However, whether these findings have relevance in the clinical context is speculative. Whether these contacts develop disease depends upon several factors of which the host immune status is prime. DNA PCR in nasal swabs of contacts of leprosy cases show positivity in 1% to 10% of cases. Of note, this may not relate directly to the prediction of disease onset.[9] Reis et al.[53] performed qPCR in blood samples from 826 household leprosy contacts and found positive PCR in 1.2% of contacts regardless of MB or PB leprosy in the index case. The contacts showed 14.78 times higher risk of developing disease after a follow-up of 7 years. IgA antibodies against natural octyl disaccharide bound to human serum albumin (NDO-HSA) (a synthetic PGL-1 compound) have been detected in serum from 63% of paucibacillary patients and in contacts of both PB and MB cases, indicating its use in follow-up of leprosy contacts.[54] A study in Brazil combined results of microscopic analysis of smears, serology for anti-LID-1 and anti-ND-O-LID, and quantitative PCR performed on slit skin smears using machine learning and the random forest system. They found the useful prediction of disease in household contacts using this algorithm.[55] The World Health Organiation (WHO) currently does not recommend any test for detecting infection in household contacts.[2]

Prognostic tests

The prognosis of leprosy cases relies heavily on the immune status of the patient and is amply defined by the rank in the Ridley Jopling scale.

SSS and skin biopsies

Microscopic assessment can guide the prognosis of an individual patient. Besides the bacteriologic and morphologic indices, which are performed on SSS, histopathologic assessment of skin or nerve biopsies is also useful. The granuloma fraction (which is a measure of the part of dermis involved by granulomas) and bacterial index of granulomas (which is similar to the logarithmic assessment of bacilli on SSS) have been considered to be useful indices on skin biopsies.[7],[56],[57] Amongst 730 multibacillary patients who were followed up for up to 10 years post completion of 12 months of MDT, relapse was found to occur only in 1.7% of cases. The relapses were limited to LL cases with a BI of 3 + or higher.[58]

Nerve biopsies

The traditional view has been to treat single nerve involvement on clinical examination as paucibacillary and multiple nerve involvement as multibacillary leprosy. However, once pure neuritic leprosy is diagnosed, attention should be paid to the specific histopathologic features for correct therapy and prognostication. A study on nerve biopsies in pure neuritic leprosy identified an appreciable number of cases showing clinical mononeuropathy with BB or LL histology and those showing involvement of multiple nerves with TT or BT on nerve biopsy. This highlights the discrepancy in clinical versus histologic subtyping and potential overtreatment of paucibacillary and under-treatment of multibacillary cases if nerve biopsies are not performed in PNL.[12] Nerve biopsies can better subtype, guide treatment, and prognosticate in pure neuritic leprosy cases.[12]

Immunologic tests

IgA antibodies have been detected in saliva from household contacts of leprosy patients. Higher the quantum of exposure to lepra bacilli, higher was the level of salivary IgA. This indicates that IgA antibodies may confer local mucosal protection against lodgement of M. leprae infection.

Susceptibility genes

Numerous genetic associations have been found with leprosy, some of which relate to susceptibility for the development of leprosy and others to the development of different clinical subtypes. The presence of HLA-DR2 has been linked to the development of leprosy as such and HLA-DRB1 to resistance for the development of Lepromatous forms of leprosy, in a study from Brazil.[59] Polymorphisms in genes involving toll-like receptors, vitamin D receptor, cytokine genes etc., may modify the host response to M. Leprae infection.[60] These are of research interest as yet but may play a role in altering the prognosis of individual patients.

Predictive tests

Predictive analyses in leprosy relate to treatment response, drug resistance prediction, and to forecasting the likelihood of reactions. Multidrug therapy (MDT) is used in leprosy with the aim of preventing drug resistance. However, infrequent cases of resistance to rifampicin alone or both rifampicin and ofloxacin both are reported from many countries.[2]

Treatment response prediction

The bacillary index differentiates paucibacillary from multibacillary leprosy and is a useful guide to the duration of MDT. It can also be used to monitor treatment, and it has been recommended to continue treatment till smear negativity.[61] The morphological index (MI) is an advancement over the BI and is a measure of solid staining (live) bacilli alone. It is assumed to predict treatment failure, relapse, and nonconformity to drug regimens amongst leprosy patients.[3] PCR for M. leprae has also been performed longitudinally in cases of chemotherapy. It was found that quantitative real-time PCR for M. Leprae hsp18mRNA showed a better correlation with treatment rather than M. leprae DNA.[62] Sixteen s rRNA which is fragile and lost from dead lepra bacilli has also been used in reverse transcriptase (RT) PCR techniques and is thought to test positive only in cases with viable bacilli. An application of this technique has been suggested to assess treatment results in leprosy cases.[63] Cytokine assays using ELISA have also been performed sequentially and found to decrease significantly post-therapy.[52]

The presence of antilipoarabinomannan in the saliva of leprosy patients has been tested and it has been suggested as a simple, inexpensive salivary ELISA for detecting treatment response.[64] The mouse footpad technique which is labor intensive and time-consuming was till late the only method to detect drug resistance in leprosy. In an extensive study across 19 countries, Cambau et al.[65] identified gene subsets using PCR sequencing which are related with drug resistance to MDT. This will pave the way for simpler cost-effective techniques for detecting drug resistance in the future. In a study from South India, rifampicin, dapsone, and ofloxacin resistance was detected using PCR gene amplification in 1.3%, 2.6%, and 6.4% of nerve biopsies from pure neuritic leprosy cases, respectively.[66] Andrade in a metanalysis found a summary sensitivity of 78% to 88% and summary specificity of 96% for molecular tests for drug resistance in leprosy.[67]

Prediction of lepra reactions

Type 1 and 2 reactions are debilitating occurrences that can have long-term sequelae if not promptly treated. Both are an immunologic phenomenon. Type 1 reactions are delayed hypersensitivity-mediated, whereas type 2 reactions involve immune complexes. Madan et al.[52] found raised levels of several cytokines in lepra reactions, of which IL10 showed significant elevation in Type 2 reaction and TNF alpha in Type 1 reaction. Serum levels of anti-PGL-1 and anti-NDO-LID-1 antibodies were also found to be raised in Type 2 reactions.[68] Whole blood RNA expression was evaluated in 1090 samples using reverse transcription multiplex ligation–dependent probe amplification, and five genes indicative of risk for lepra reactions were identified. It has been claimed that reversal reactions in leprosy can be predicted using this genetic signature up to 2 weeks before clinical symptoms appear. Though currently of research interest, this may help the prevention of permanent disability associated with reactions.[69]

In a study using immunohistochemistry on skin biopsies from reactive and nonreactive leprosy patients, it was noted that expressions of cyclooxygenase 2 (COX 2) and vascular endothelial growth factor (VEGF) showed significant difference. The levels were highest in Type 1 reactions, followed by type 2 reactions and lowest in non-reactive leprosy cases. As inhibitor drugs for COX 2 and VEGF are available, these may be considered in the specific treatment of reactive leprosy.[70]


   Conclusion Top


Leprosy elimination is the WHO global strategy for 2016–2020 - “Accelerating towards a leprosy-free world.”[71] The cornerstone of this program is improved diagnosis of symptomatic and asymptomatic leprosy. The WHO currently recommends clinical examination along with SSS or biopsy as the recommended methods for leprosy diagnosis. Although slit skin smears and biopsies are available at most centers in India, leprosy PCR, serum ELISA, and cytokine profile testing are limited to research use currently and are available only at tertiary care centers in India.[42],[49] The ideal test would be one which is accurate, reliable, inexpensive, able to detect all subtypes of leprosy, requires little technical expertise, and is applicable to field conditions. We have moved forward in the field of leprosy diagnostics with the use of blood and saliva as noninvasive samples with molecular tests showing enhanced sensitivity. We suggest an algorithm for optimal diagnosis (utilizing the available tests) in cases with skin or neurologic manifestations clinically suspicious of leprosy, and their response prediction and prognostication postdiagnosis. [Figure 2] Such an approach would be ideal in resource unconstrained settings. However, some pitfalls remain. Though many tests show appreciable results in MB cases, PB cases are difficult to diagnose both clinically and using any of the available tests –SSS, ELISA, lateral flow assays, or PCR. PCR additionally suffers from requiring technical expertise and being expensive. Commercial leprosy PCR kits are not freely available. [Table 2] compares the characteristics and utility of available tests for leprosy. [Table 2] In the absence of a test aiming at detection of infection in asymptomatic patients, larger optimally planned studies are required to define tests that would be effective in diagnosing leprosy cases across the immune spectrum. Longitudinal assays to detect the natural course in infected contacts and tests measuring both cellular and humoral immune responses hold promise for the future.
Table 2: Comparison of various tests available for diagnosis of Leprosy

Click here to view
Figure 2: Approach to a case of suspected leprosy

Click here to view


Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
mondiale de la Santé O, World Health Organization. Global leprosy (Hansen disease) update, 2019: time to step-up prevention initiatives–Situation de la lèpre (maladie de Hansen) dans le monde, 2019: le moment est venu d'intensifier les initiatives de prévention. Weekly Epidemiological Record= Relevé épidémiologique hebdomadaire. 2020;95:417-38.  Back to cited text no. 1
    
2.
World Health Organization (WHO). Guidelines for the Diagnosis, Treatment and Prevention of Leprosy. New Delhi: World Health Organization, Regional Office for South-East Asia; 2017. Available from: https://www.who.int/lep/resources/9789290226383/en/. [Last accessed on 2021 Oct 20].  Back to cited text no. 2
    
3.
Mahajan VK. Slit-skin smear in leprosy: Lest we forget it! Indian J Lepr 2013;85:177-83.  Back to cited text no. 3
    
4.
Ridley DS, Jopling WH. Classification of leprosy according to immunity; a five-group system. Int J Lepr 1966;34:255-73.  Back to cited text no. 4
    
5.
Lyon S, Lyon AC, Da Silva RC, Grossi MA, Lyon SH, Bührer-Sékula S, et al. A comparison of ML Flow serology and slit skin smears to assess the bacterial load in newly diagnosed leprosy patients in Brazil. Lepr Rev 2008;79:162-70.  Back to cited text no. 5
    
6.
Banerjee S, Biswas N, Kanti Das N, Sil A, Ghosh P, Hasanoor Raja AH, et al. Diagnosing leprosy: Revisiting the role of the slit-skin smear with critical analysis of the applicability of polymerase chain reaction in diagnosis. Int J Dermatol 2011;50:1522-7.  Back to cited text no. 6
    
7.
Massone C, Belachew WA, Schettini A. Histopathology of the lepromatous skin biopsy. Clin Dermatol 2015;33:38-45.  Back to cited text no. 7
    
8.
Massone C. Histopathology of the skin. In: Nunzi E, Massone C, editors. Leprosy, A Practical Guide. Berlin: Springer; 2012. p. 115-36.  Back to cited text no. 8
    
9.
Maymone MB, Laughter M, Venkatesh S, Dacso MM, Rao PN, Stryjewska BM, et al. Leprosy: Clinical aspects and diagnostic techniques. J Am Acad Dermatol 2020;83:1-14.  Back to cited text no. 9
    
10.
Malhotra KP, Suvirya S, Malhotra HS, Kumar B, Gupta A. Does histoid leprosy represent a locally hyperimmune variant of lepromatous leprosy? QJM 2019;112:429-35.  Back to cited text no. 10
    
11.
Suvirya S, Pathania S, Malhotra KP, Jain A, Verma P, Kumari P. A case of diffuse lepromatous leprosy with Lucio phenomenon. QJM 2020;113:138-9.  Back to cited text no. 11
    
12.
Kulshreshtha D, Malhotra KP, Malhotra HS, Thacker AK, Garg RK, Singh AK, et al. Mandating nerve biopsy: A step towards personalizing therapy in pure neuritic leprosy. J Peripher Nerv Syst 2018;23:190-6.  Back to cited text no. 12
    
13.
Nagappa M, Chickabasaviah YT, Mahadevan A, Parthiban R, Taly AB. Pitfalls in the diagnosis of leprous neuropathy: Lessons learnt from a University hospital in an endemic zone. J Neurol Sci 2015;357:252-6.  Back to cited text no. 13
    
14.
Hui M, Uppin MS, Challa S, Meena AK, Kaul S. Pure neuritic leprosy: Resolving diagnostic issues in acid fast bacilli (AFB)-negative nerve biopsies: A single Centre experience from South India. Ann Indian Acad Neurol 2015;18:292-7.  Back to cited text no. 14
[PUBMED]  [Full text]  
15.
Antunes SL, Chimelli L, Jardim MR, Vital RT, Nery JA, Corte-Real S, et al. Histopathological examination of nerve samples from pure neural leprosy patients: Obtaining maximum information to improve diagnostic efficiency. Mem Inst Oswaldo Cruz 2012;107:246-53.  Back to cited text no. 15
    
16.
Jacob M, Mathai R. Diagnostic efficacy of cutaneous nerve biopsy in primary neuritic leprosy. Int J Lepr 1988;56:56-60.  Back to cited text no. 16
    
17.
Kulshreshtha D, Malhotra KP, Maurya PK, Singh AK, Thacker AK, Husain N. Nerve biopsy in Indian patients with mononeuropathy multiplex of undetermined etiology. Muscle Nerve 2017;55:23–7.  Back to cited text no. 17
    
18.
Jain S, Visser LH, Suneetha S. Imaging techniques in leprosy clinics. Clin Dermatol 2016;34:70-8.  Back to cited text no. 18
    
19.
Shukla B, Verma R, Kumar V, Kumar M, Malhotra KP, Garg RK, et al. Pathological, ultrasonographic, and electrophysiological characterization of clinically diagnosed cases of pure neuritic leprosy. J Peripher Nerv Syst 2020;25:191-203.  Back to cited text no. 19
    
20.
Martinoli C, Derchi LE, Bertolotto M, Gandolfo N, Bianchi S, Fiallo P, et al. US and MR imaging of peripheral nerves in leprosy. Skeletal Radiol 2000;29:142-50.  Back to cited text no. 20
    
21.
Maeda SM, Rotta O, Michalany NS, Camargo ZP, Sunderkötter C, Tomimori-Yamashita J. Comparison between anti-PGL-I serology and Mitsuda reaction: Clinical reading, microscopic findings and immunohistochemical analysis. Lepr Rev 2003;74:263-74.  Back to cited text no. 21
    
22.
Araujo S, Rezende MM, Sousa DC, Rosa MR, Santos DC, Goulart LR, et al. Risk-benefit assessment of Bacillus Calmette-Guérin vaccination, anti-phenolic glycolipid I serology, and Mitsuda test response: 10-year follow-up of household contacts of leprosy patients. Rev Soc Bras Med Trop 2015;48:739-45.  Back to cited text no. 22
    
23.
Sengupta U. Recent laboratory advances in diagnostics and monitoring response to treatment in leprosy. Indian Dermatol Online J 2019;10:106-14.  Back to cited text no. 23
[PUBMED]  [Full text]  
24.
Vissa VD, Brennan PJ. The genome of Mycobacterium leprae: A minimal mycobacterial gene set. Genome Biol 2001;2:REVIEWS1023.  Back to cited text no. 24
    
25.
Rodrigues LC, Lockwood DNj. Leprosy now: Epidemiology, progress, challenges, and research gaps. Lancet Infect Dis 2011;11:464-70. doi: 10.1186/gb-2001-2-8-reviews1023.  Back to cited text no. 25
    
26.
Gurung P, Gomes CM, Vernal S, Leeflang MM. Diagnostic accuracy of tests for leprosy: A systematic review and meta-analysis. Clin Microbiol Infect 2019;25:1315-27.  Back to cited text no. 26
    
27.
Gama RS, Leite LA, Colombo LT, Fraga LA. Prospects for new leprosy diagnostic tools, a narrative review considering ELISA and PCR assays. Rev Soc Bras Med Trop 2020;53:e20200197.  Back to cited text no. 27
    
28.
Izumi S, Fujiwara T, Ikeda M, Nishimura Y, Sugiyama K, Kawatsu K. Novel gelatin particle agglutination test for serodiagnosis of leprosy in the field. J Clin Microbiol 1990;28:525-9.  Back to cited text no. 28
    
29.
Bührer-Sékula S, Cunha MG, Foss NT, Oskam L, Faber WR, Klatser PR. A simple dipstick assay for the detection of antibodies to phenolic glycolipid-I of Mycobacterium leprae. Am J Trop Med Hyg 1998;58:133-6.  Back to cited text no. 29
    
30.
Duthie MS, Balagon MF, Maghanoy A, Orcullo FM, Cang M, Dias RF, et al. Rapid quantitative serological test for detection of infection with Mycobacterium leprae, the causative agent of leprosy. J Clin Microbiol 2014;52:613-9.  Back to cited text no. 30
    
31.
van Hooij A, Geluk A. In search of biomarkers for leprosy by unraveling the host immune response to Mycobacterium leprae. Immunol Rev 2021;301:175-92.  Back to cited text no. 31
    
32.
Moura RS, Calado KL, Oliveira ML, Bührer-Sékula S. Leprosy serology using PGL-I: A systematic review. Rev Soc Bras Med Trop 2008;41(Suppl 2):11-8.  Back to cited text no. 32
    
33.
Araújo S, Lobato J, Reis Ede M, Souza DO, Gonçalves MA, Costa AV, et al. Unveiling healthy carriers and subclinical infections among household contacts of leprosy patients who play potential roles in the disease chain of transmission. Mem Inst Oswaldo Cruz 2012;107(Suppl 1):55-9.  Back to cited text no. 33
    
34.
Douglas JT, Cellona RV, Fajardo TT Jr, Abalos RM, Balagon MV, Klatser PR. Prospective study of serological conversion as a risk factor for development of leprosy among household contacts. Clin Diagn Lab Immunol 2004;11:897-900.  Back to cited text no. 34
    
35.
Santos DF, Mendonça MR, Antunes DE, Sabino EF, Pereira RC, Goulart LR, et al. Revisiting primary neural leprosy: Clinical, serological, molecular, and neurophysiological aspects. PLoS Negl Trop Dis 2017;11:e0006086.  Back to cited text no. 35
    
36.
Martinez AN, Talhari C, Moraes MO, Talhari S. PCR-based techniques for leprosy diagnosis: From the laboratory to the clinic. PLoS Negl Trop Dis 2014;8:e2655.  Back to cited text no. 36
    
37.
Tatipally S, Srikantam A, Kasetty S. Polymerase Chain Reaction (PCR) as a potential point of care laboratory test for leprosy diagnosis-A systematic review. Trop Med Infect Dis 2018;3:107.  Back to cited text no. 37
    
38.
Azevedo MC, Ramuno NM, Fachin LR, Tassa M, Rosa PS, Belone AF, et al. qPCR detection of Mycobacterium leprae in biopsies and slit skin smear of different leprosy clinical forms. Braz J Infect Dis 2017;21:71-8.  Back to cited text no. 38
    
39.
Torres RT, Fachi MM, Böger B, Marson BM, Ferreira VL, Pontarolo R, et al. Sensitivity and specificity of multibacillary and paucibacillary leprosy laboratory tests: A systematic review and meta-analysis. Diagn Microbiol Infect Dis 2021;100:115337.  Back to cited text no. 39
    
40.
Cheng X, Sun L, Zhao Q, Mi Z, Yu G, Wang Z, et al. Development and evaluation of a droplet digital PCR assay for the diagnosis of paucibacillary leprosy in skin biopsy specimens. PLoS Negl Trop Dis 2019;13:e0007284.  Back to cited text no. 40
    
41.
Chaitanya VS, Cuello L, Das M, Sudharsan A, Ganesan P, Kanmani K, et al. Analysis of a novel multiplex polymerase chain reaction assay as a sensitive tool for the diagnosis of indeterminate and tuberculoid forms of leprosy. Int J Mycobacteriol 2017;6:1-8.  Back to cited text no. 41
[PUBMED]  [Full text]  
42.
Tiwari V, Malhotra K, Khan K, Maurya PK, Singh AK, Thacker AK, et al. Evaluation of polymerase chain reaction in nerve biopsy specimens of patients with Hansen's disease. J Neurol Sci 2017;380:187-90.  Back to cited text no. 42
    
43.
Siwakoti S, Rai K, Bhattarai NR, Agarwal S, Khanal B. Evaluation of Polymerase Chain Reaction (PCR) with Slit Skin Smear Examination (SSS) to confirm clinical diagnosis of leprosy in Eastern Nepal. PLoS Negl Trop Dis 2016;10:e0005220.  Back to cited text no. 43
    
44.
de Wit MY, Douglas JT, McFadden J, Klatser PR. Polymerase chain reaction for detection of Mycobacterium leprae in nasal swab specimens. J Clin Microbiol 1993;31:502-6.  Back to cited text no. 44
    
45.
Patrocínio LG, Goulart IM, Goulart LR, Patrocínio JA, Ferreira FR, Fleury RN. Detection of Mycobacterium leprae in nasal mucosa biopsies by the polymerase chain reaction. FEMS Immunol Med Microbiol 2005;44:311-6.  Back to cited text no. 45
    
46.
Wen Y, Xing Y, Yuan LC, Liu J, Zhang Y, Li HY. Whole-blood nested-PCR amplification of M. leprae-specific DNA for early diagnosis of leprosy. Am J Trop Med Hyg 2013;88:918-22.  Back to cited text no. 46
    
47.
Caleffi KR, Hirata RD, Hirata MH, Caleffi ER, Siqueira VL, Cardoso RF. Use of the polymerase chain reaction to detect Mycobacterium leprae in urine. Braz J Med Biol Res 2012;45:153-7.  Back to cited text no. 47
    
48.
Chen X, Xing Y, He J, Tan F, You Y, Wen Y. Develop and field evolution of single tube nested PCR, SYBRGreen PCR methods, for the diagnosis of leprosy in paraffin-embedded formalin fixed tissues in Yunnan Province, a hyper endemic area of leprosy in China. PLoS Negl Trop Dis 2019;13:e0007731.  Back to cited text no. 48
    
49.
Vengalil S, Lavania M, Singh I, Nashi S, Preethish-Kumar V, Polavarapu K, et al. Appropriately selected nerve in suspected leprous neuropathy yields high positive results for mycobacterium leprae DNA by polymerase chain reaction method. Am J Trop Med Hyg 2020;103:209-13.  Back to cited text no. 49
    
50.
Geluk A, Bobosha K, van der Ploeg-van Schip JJ, Spencer JS, Banu S, Martins MV, et al. New biomarkers with relevance to leprosy diagnosis applicable in areas hyperendemic for leprosy. J Immunol 2012;188:4782-91.  Back to cited text no. 50
    
51.
Yamamura M, Uyemura K, Deans RJ, Weinberg K, Rea TH, Bloom BR, et al. Defining protective responses to pathogens: Cytokine profiles in leprosy lesions. Science 1991;254:277-9.  Back to cited text no. 51
    
52.
Madan NK, Agarwal K, Chander R. Serum cytokine profile in leprosy and its correlation with clinico-histopathological profile. Lepr Rev 2011;82:371-82.  Back to cited text no. 52
    
53.
Reis EM, Araujo S, Lobato J, Neves AF, Costa AV, Gonçalves MA, et al. Mycobacterium leprae DNA in peripheral blood may indicate a bacilli migration route and high-risk for leprosy onset. Clin Microbiol Infect 2014;20:447-52.  Back to cited text no. 53
    
54.
Silva KK, de Oliveira EE, Elias CM, Pereira IE, Pinheiro RO, Sarno EN, et al. Serum IgA antibodies specific to M. leprae antigens as biomarkers for leprosy detection and household contact tracking. Front Med (Lausanne) 2021;8:698495.  Back to cited text no. 54
    
55.
Gama RS, Souza ML, Sarno EN, Moraes MO, Gonçalves A, Stefani MM, et al. A novel integrated molecular and serological analysis method to predict new cases of leprosy amongst household contacts. PLoS Negl Trop Dis 2019;13:e0007400.  Back to cited text no. 55
    
56.
Bhushan P, Sardana K, Koranne RV, Choudhary M, Manjul P. Diagnosing multibacillary leprosy: A comparative evaluation of diagnostic accuracy of slit-skin smear, bacterial index of granuloma and WHO operational classification. Indian J Dermatol Venereol Leprol 2008;74:322-6.  Back to cited text no. 56
[PUBMED]  [Full text]  
57.
Srinivas D, Rao PN, Lakshmi TS, Suneetha S. Bacterial index of granuloma and its relevance compared to BI of skin smears. Lepr Rev 2002;73:79-80.  Back to cited text no. 57
    
58.
Dogra S, Kumaran MS, Narang T, Radotra BD, Kumar B. Clinical characteristics and outcome in multibacillary (MB) leprosy patients treated with 12 months WHO MDT-MBR: A retrospective analysis of 730 patients from a leprosy clinic at a tertiary care hospital of Northern India. Lepr Rev 2013;84:65-75.  Back to cited text no. 58
    
59.
da Silva SA, Mazini PS, Reis PG, Sell AM, Tsuneto LT, Peixoto PR, et al. HLA-DR and HLA-DQ alleles in patients from the south of Brazil: Markers for leprosy susceptibility and resistance. BMC Infect Dis 2009;9:134.  Back to cited text no. 59
    
60.
Mazini PS, Alves HV, Reis PG, Lopes AP, Sell AM, Santos-Rosa M, et al. Gene association with leprosy: A review of published data. Front Immunol 2016;6:658.  Back to cited text no. 60
    
61.
Girdhar BK, Girdhar A, Kumar A. Relapses in multibacillary leprosy patients: Effect of length of therapy. Lepr Rev 2000;71:144-53.  Back to cited text no. 61
    
62.
Lini N, Shankernarayan NP, Dharmalingam K. Quantitative real-time PCR analysis of Mycobacterium leprae DNA and mRNA in human biopsy material from leprosy and reactional cases. J Med Microbiol 2009;58:753-9.  Back to cited text no. 62
    
63.
Kurabachew M, Wondimu A, Ryon JJ. Reverse transcription-PCR detection of Mycobacterium leprae in clinical specimens. J Clin Microbiol 1998;36:1352-6.  Back to cited text no. 63
    
64.
Nahas AA, Lima MI, Goulart IM, Goulart LR. Anti-lipoarabinomannan-specific salivary IgA as prognostic marker for leprosy reactions in patients and cellular immunity in contacts. Front Immunol 2018;9:1205.  Back to cited text no. 64
    
65.
Cambau E, Saunderson P, Matsuoka M, Cole ST, Kai M, Suffys P, et al. WHO surveillance network of antimicrobial resistance in leprosy. Antimicrobial resistance in leprosy: Results of the first prospective open survey conducted by a WHO surveillance network for the period 2009-15. Clin Microbiol Infect 2018;24:1305-10.  Back to cited text no. 65
    
66.
Mahajan NP, Lavania M, Singh I, Nashi S, Preethish-Kumar V, Vengalil S, et al. Evidence for mycobacterium leprae drug resistance in a large cohort of leprous neuropathy patients from India. Am J Trop Med Hyg 2020;102:547-52.  Back to cited text no. 66
    
67.
Andrade ES, Brandão JG, da Silva JS, Kurizky PS, Rosa PS, de Araújo WN, et al. A systematic review and meta-analysis of studies on the diagnostic accuracy and screening of tests to detect antimicrobial resistance in leprosy. Diagn Microbiol Infect Dis 2021;100:115325.  Back to cited text no. 67
    
68.
Devides AC, Rosa PS, de Faria Fernandes Belone A, Coelho NM, Ura S, Silva EA. Can anti-PGL-1 and anti-NDO-LID-1 antibody titers be used to predict the risk of reactions in leprosy patients? Diagn Microbiol Infect Dis 2018;91:260-5.  Back to cited text no. 68
    
69.
Tió-Coma M, van Hooij A, Bobosha K, van der Ploeg-van Schip JJ, Banu S, Khadge S, et al. Whole blood RNA signatures in leprosy patients identify reversal reactions before clinical onset: A prospective, multicenter study. Sci Rep 2019;9:17931.  Back to cited text no. 69
    
70.
Malhotra KP, Suvirya S, Malhotra HS, Kumar B, Kumar S, Husain N. Cyclooxygenase 2 and vascular endothelial growth factor-potential targets to manage lepra reactions: A case-control study. Dermatol Ther 2021;34:e14882.  Back to cited text no. 70
    
71.
World Health Organization, Regional Office for South-East Asia, Global Leprosy Programme. Global Leprosy Strategy 2016–2020: Accelerating Towards a Leprosy-Free World. New Delhi: WHO Regional Office for South-East Asia; 2016. Available from: http://apps.who.int/iris/bitstream/handle/10665/208824/9789290225096_en.pdf.  Back to cited text no. 71
    

Top
Correspondence Address:
Kiran Preet Malhotra
Department of Pathology, Dr. Ram Manohar Lohia Institute of Medical Sciences, Gomti Nagar, Lucknow - 226 010, Uttar Pradesh
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijpm.ijpm_1083_21

Rights and Permissions


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2]



 

Top
 
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Email Alert *
    Add to My List *
* Registration required (free)  


    Abstract
   Introduction
   Conclusion
    References
    Article Figures
    Article Tables

 Article Access Statistics
    Viewed182    
    Printed6    
    Emailed0    
    PDF Downloaded15    
    Comments [Add]    

Recommend this journal