|Year : 2019 | Volume
| Issue : 2 | Page : 106-114
Recent laboratory advances in diagnostics and monitoring response to treatment in leprosy
Departemnt of Microbiology and Immunology, Stanley Browne Laboratory, TLM Community Hospital, Nand Nagri, New Delhi, India
|Date of Web Publication||15-Mar-2019|
Stanley Browne Laboratory, TLM Community Hospital, Nand Nagri, New Delhi - 110 093
Source of Support: None, Conflict of Interest: None
| Abstract|| |
The present review briefly summarizes the highlights of the recent advances in Mycobacterium leprae-specific tests for early diagnosis of leprosy. In addition to establishing the diagnosis of clinical cases of leprosy, these tests have also been used to detect subclinical infections in endemic population. Several attempts have been made from 1980 onward for standardization of specific diagnostic assays for early detection of leprosy. Brief account about the development and use of these assays has been described in this review article.
Keywords: Antigens, leprosy, Mycobacterium leprae, polymerized chain reaction, serology
|How to cite this article:|
Sengupta U. Recent laboratory advances in diagnostics and monitoring response to treatment in leprosy. Indian Dermatol Online J 2019;10:106-14
|How to cite this URL:|
Sengupta U. Recent laboratory advances in diagnostics and monitoring response to treatment in leprosy. Indian Dermatol Online J [serial online] 2019 [cited 2019 Jun 18];10:106-14. Available from: http://www.idoj.in/text.asp?2019/10/2/106/254292
| Introduction|| |
Leprosy, a chronic infectious disease of skin and neurons, caused by Mycobacterium leprae (ML) and Mycobacterium lepromatosis, has been known to inflict humans from ancient times. It is still prevalent in many countries including India. India still hosts 63% of the world leprosy population, and more than 70% new cases of leprosy in the world are detected in India every year. Although India attained the elimination figure, a prevalence rate (PR) of less than 1 case per 10,000 population size (<0.9/10,000) in December 2005, but PR is still persisting at 0.74/10,000 (April 2017) indicating no significant decline in PR over the last decade. Furthermore ANCDR (annual new case detection rate) which had almost plateaued earlier, has demonstrated a rising trend recently (from 9.71/100,000 of 2016 to 10.12/100,000 in 2017). All these trends indicate that despite the advent of multidrug therapy (MDT), burden and transmission of leprosy in India is still a matter of major health concern. In order to limit the burden and transmission of leprosy in the community, early detection and treatment is of utmost importance.
Leprosy is clinically diagnosed on the basis of presence of following cardinal signs: (i) hypopigmented or erythematous anesthetic patch on skin, (ii) thickened and/or tender peripheral or cutaneous nerve supplying the affected area, and (iii) acid fast bacilli in the skin smear. The disease manifests as spectrum of different clinical forms determined by the immune status of the host. This spectrum ranges from tuberculoid (TT) and borderline tuberculoid (BT) in patients having a strong cell-mediated immunity (CMI) and weak humoral immunity (HI) to borderline lepromatous (BL) and lepromatous (LL) forms in those with a robust HI and an almost nonexistent CMI to M. leprae with mid-borderline (BB) form lying in between. The spectrum of clinical manifestation has also been classified on an immunohistological and bacteriological scale by Ridley and Jopling. In addition, there is a very early form of disease termed as indeterminate (I) leprosy which appears with small hypopigmented macules in skin without any loss of sensation. If it is left untreated, it may progress to other clinical forms or clears on its own owing to upgradation in CMI. Another form of leprosy which has been encountered frequently, remains confined to single or multiple nerves without involving the skin is termed as pure neuritic leprosy. Histopathological features of the involved nerves in pure neuritic leprosy is similar to that of involved skin in other forms of lprosy. Diagnosis of leprosy thus can be made by the clinical signs alone; however, in absence of definitive cardinal features, confirmation of leprosy can be difficult in some patients especially in a non-endemic country. Histopathology is the usual modality for confirmation of a clinically doubtful case of leprosy. However, other procedures like skin testing with M. leprae antigen (lepromin), antibody responses of the host to M. leprae and molecular techniques to detect the components of M. leprae in the lesions have also been used for diagnosis of leprosy at early stage. The present review discusses in detail the various approaches which have been developed and adopted for clinical diagnosis and monitoring of response in on-treatment leprosy cases.
| Use of Lepromin|| |
Lepromin is a saline suspension of whole M. leprae, which is inoculated intradermally on the volar surface of forearm to test the status of delayed type of hypersensitivity (DTH) response or CMI of an individual to the organism. While patients at the TT/BT end evoke a strong DTH skin reaction, those at the BL/LL end, fail to develop any skin reaction to lepromin. Later, M. leprae soluble antigens were prepared either by disruption or sonication of purified armadillo derived M. leprae and these soluble antigens of M. leprae also known as “leprosin” showed a background sensitization pattern of the population to mycobacterial antigens with bimodal distribution like lepromin. These antigens have been better utilized for classification of leprosy and for evaluation of CMI of leprosy patients on treatment. As M. leprae have common sharing antigens with other environmental mycobacteria which are ubiquitous in the nature. lepromin or leprosin test positivity will only indicate the status of CMI of the individual to M. leprae or cross-reactive mycobacterial antigens. As the positivity of these tests are not specific for M. leprae infection, lepromin or “leprosin” can't be used for diagnosis of leprosy. However, negative response to lepromin in a subject will indicate deficiency in host CMI to M. leprae and the individual may benefit from immunomodulation to boost the CMI to M. leprae. Therefore, there is a scope for lepromin to be used for mass survey to identify the prospective lepromin-negative candidates in a population who can benefit from vaccines to M. leprae.
Use of M. leprae–Specific Serology
Leprosy-specific serological tests emerged only after identification of M. leprae-specific antigens. Specificity and sensitivity of the serological assays have been summarized in [Table 1].
|Table 1:Comparative efficacies of immunological and molecular markers in diagnosis of leprosy|
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Serological test using phenolic glycolipid-1
Phenolic glycolipid-1 (PGL-1) is one of the first mycobacterial antigens which was identified and isolated from the major glycolipid cell wall antigen of the bacterium. Using this as an antigen, an enzyme-linked immunosorbent assay (ELISA) was developed initially for diagnosis of leprosy.,, Although the sensitivity of this assay was 90-95% in BL/LL cases; the sensitivity was poor for detection of PB cases, (0-40%). Healthy endemic controls tested mostly negative in this assay for PGL-1 antibody and about 26% of household contacts were found to be positive. Later on, trisccharride [3,6-di-O-methyl-β-d-glucopyranosyl-(1→4)-2,3-di-O-methyl-α-l-rhamnopyranosyl-(1→2)-3-O-methyl-α-l-rhamnopyranose],, and the disaccharide components of PGL-1 were found to be the components which react specifically with IgM antibodies in patients' sera.. Hence, these synthetic sugars, natural trisaccharide (NT), and natural disaccharide (ND) were synthesized individually and conjugated with either bovine serum albumin (BSA) or human serum albumin (HSA) using either octyl (O) or phenyl (P) linker arms (ND-O-BSA/HSA or NT-O-BSA/NT-P-BSA) and used in standardization of ELISA for diagnosis of leprosy., It was noted that these glycoconjugates had higher affinity for IgM antibody than PGL-1 and showed a rising trend in the antibody levels from tuberculoid to lepromatous spectrum associated with increase in bacterial load.,,, However, a positive correlation with bacterial load and PGL-1 antibody levels was not always observed. Using this neoglycoconjugate, newer assays known as M. leprae dipstick assay, and particle agglutination assay were developed. In dipstick format, two antigen bands are present, one of which signifies reactivity to ND-O-BSA and the other is used as internal control for human IgM. The agreement of dipstick assay with ND-O-BSA-based assay was found to be 94.9%. The other particle agglutination assay, gelatin particle agglutination test, was developed by the initial activation of colored gelatin particles by tannic acid and finally mixing with NT-P-BSA. This NT-P-BSA-labeled gelatin particles agglutinated with serial two-fold dilutions of patientsera with an average cutoff value for positivity ranging between serum dilutions of 1:64 and 1:128.
In order to make the ML-dipstick assay suitable for field conditions, the neoglycoconjugate-based assay was modified by developeing it on a solid support using immunochromatographic technique in a lateral flow assay, termed as ML-flow test. In this assay, the nitrocellulose (NC) strips are loaded with 1-mm wide parallel lines of human IgM (positive control) and neoglycoconjugate, which react with the IgM antibody present in patientserum. The NC strip is encased in a plastic module with a sample charging slot and is followed by a reagent pad area for serum or whole blood sample with diluents to flow through and to be absorbed in the absorbent pad at the bottom of the case. Samples while flowing through the reagent pad pick up the colloidal gold-labeled antihuman IgM which binds specifically human IgM present on the parallel lines to give positive results for the test and IgM. The test is read generally within 10 min of charging of the samples. The specificity of the ML-flow test was found to be 90.2%. The agreement between ML-flow test and PGL-1 ELISA was found to be 91%. In addition, this test was found to be positive in 97.4% of multibacillary (MB) and 40% of paucibacillary (PB) cases and in 28.6% of household contacts. However, it was noted that in highly endemic countries, about 10% of uninfected individuals may be positive to PGLantibody.,
Status of PGL-1 antibody level with treatment
As antibody levels have been found to correlate with the bacterial load,,,, it is justified to presume that the levels of PGL-1 antibody will decline after adequate chemotherapy. Hence PGL-1 antibody-based serology could provide a method to monitor leprosy patients under treatment. A recent cohort study with 105 leprosy patients on MDT regimen, followed up for 6 months to 2 years with periodic assessment of BI and PGL-1antibody levels, showed that monitoring of antibody levels in leprosy patients while on chemotherapy is useful in determining the efficacy of MDT. Several other earlier studies also showed significant reduction in the levels of PGL-1 antibody after chemotherapy in leprosy patients.,,,,,
Using immuno-biochemical technique, the evidence for the presence of 35kD protein in the membrane of M. leprae was established and the monoclonal antibody, MLO3-A1, reacted specifically with the epitope on 35kD antigen of ML. After identification of the gene encoding 35kD of ML, it could be cloned in Mycobacterium smegmatis and was available in sufficient quantities in pure form as recombinant 35kD (r35kD). It was revealed later that 82% and 90% DNA and amino acids, respectively, of ML 35kD are shared with another mycobacterial species Mycobacterium avium. Another series of monoclonal antibody (MLO4) having specificity for the same 35kD was also utilized for serological studies. The assay was initially developed as a radioimmunoassay based on competitive inhibition between patient's serum and I125 labeled MLO4 and later was standardized as ELISA using horse radish peroxidase labeledMLO4. Screening of a large number of blood samples of MB and PB patients with 35kD ELISA demonstrated a sensitivity of 98.5% and 46.7%, respectively., A filter paper-based sample collection of blood from remote field area was standardized to perform field-based studies. Inspite of 35kD antigen's sharing of some genes with M. avium, Mycobacterium kansasii, and Mycobacterium paratuberculosis, the standardized serodiagnostic assay was found to be 97.5% specific and 90% sensitive in the diagnosis of leprosy. Another study which compared PGL-1-based ELISA with 35kDa-based serology, found both the assays to be reproducible and comparable. Roche et al. compared PGL-1-based ELISA and 35-kD inhibition-based ELISA for their accuracy in diagnosis of leprosy with different levels of antibodies. It was noted that while PGL-1-based ELISA was suitable for diagnosis of cases with all the levels of antibodies, 35-kD inhibition-based ELISA did not perform well for diagnosis of patients having antibody levels near the cutoff value. Later, r35kD was used directly for assessment of specificity and sensitivity. It was noted that while the specificity of the assay was 94.3%, the sensitivity for diagnosis of MB and PB cases were 83.0% and 17.0%, respectively. The reason for low sensitivity of the assay with r35kD antigen may be due to the presence of cross-reactive mycobacterial proteins of M. smegmatis in the cloned purified recombinant protein or due to the presence of subclinical infection in the exposed contacts. Further, using both PGL-1 and r35kD, a dipstick ELISA was developed and compared with the conventional ELISA, and it was noted that there was a good concordance between the dipstick and conventional ELISA. A 35kD- test card identified 59% of untreated PB cases compared to that of 27% detection by PGL-1; however, the sensitivity was found to be 90% by the r35kDtest card and 100% by PGL-1 dipstick.
Status of 35kD antibody level with treatment
The number of anesthetic patches in patients has been shown to positively correlate with the level of antibody., Later, the antibody levels were also found to correlate positively with the number of nerves involved in primary neuritic leprosy. Attempts were also made to find out the presence of antibody in urine, cerebrospinal fluid and skin scraping samples of patients.,, However, these samples were not superior to blood samples in diagnosing a case of leprosy. Antibody level against 35kD was found to decline following effective chemotherapy of patients.
Search for new antibody reactive M. leprae recombinant proteins and development of LID-1 and NDO-LID rapid test
Considering a low level of false positivity with PGL-1 antigen, a large panel of expressed recombinant proteins was analyzed in a protein array format for their reactivity with categorized leprosy sera. Antigens, which reacted strongly with patients' sera and minimally with control sera, were selected for further analysis. The proteins selected were ML0405 and ML2331, which were found to be suitable for diagnosis of MB leprosy. These two proteins have been made as a fusion construct and have been named as LID-1 [Leprosy Infectious Disease Research Institute Diagnostic-1]. LID-1 has been shown to detect particularly MB cases in Brazil, China, Japan, and Philippines.,,, As PGL-1 or ND-O-BSA/HSA conjugate assay demonstrated positive results sometimes in uninfected controls as well,,,, LID-1 assay has been preferred for diagnosis of MB leprosy. Both of these antigens LID-1 and ND-O-BSA have been synthetically conjugated to work in one platform and a rapid test based on NDO-LID has been developed and has been named as NDO-LID rapid test (Orange Life®, Rio de Janeiro, Brazil). NDO-LID kit is a ready-to-use kit for testing in field. Serum sample (10 μl) and running buffer (100 μl) are charged in the sample well causing the migration of sample and colloidal gold beads loaded with anti-IgG and anti-IgM through the membrane across the detection window. The reaction of the test and control yields a red color. Readings are recorded within 20 min of charging of samples. A clear development of the control line validates the test. A positive result is established when both the lines of control and test are developed. Visual reading scores are graded as 1+, 1.5+, and 2+ and development of a faint color or no color is considered as negative. For field application, a “point-of-care” assay was developed using a smart phone reader to record the density of color development. These rapid tests detected higher proportion of leprosy cases compared to that of laboratory-based PGL-ELISA. Using this NDO-LID rapid test for MB cases, the positivity of PGL-ELISA was enhanced from 83.3% to 87% and for PB cases from 15.4% to 21.2%. The sensitivity and specificity of NDO-LID test were found to be 87% and 96.1%, respectively, in detection of MB cases. Recently, while screening a Venezuelan MB population, no difference has been noted between the percentage of serological positivity using NDO-HSA, LID-1, and NDO-LID, although small sample size in the study could have the reason of the above observation. Screening of household contact endemic normal population revealed that the frequency of anti-NDO-LID and anti-NDO-HSA positivity was much higher in general population than that of household contacts indicating subclinical infection or exposure of the community to the infection.
Status of LID-1/NDO-LID antibody levels with treatment
It was noted that antibody level to LID-1 declined more rapidly after MDT regimen compared to that of PGL-1-antibody level. Recently, in a study which detected antibody levels using all the three antigens PGL-1, LID-1, and NDO-LID, found that the antibody levels declined significantly after 6 months of uniform MDT (UMDT) or 12 months of full course of MDT. This reduction in antibody levels also correlated with reduction in bacillary load. Further, this group suggested that UMDT was noted to be similar to full course MDT in reduction of both the antibody levels and bacillary burden.
Use of M. leprae-specific molecule employing polymerized chain reaction
M. leprae -specific polymerized chain reaction (PCR) can be routinely performed with a variety of biological specimens like skin biopsies, skin sections, skin smears, nerve sections, biological fluids such as blood, pleural effusions, ascetic fluid, cerebrospinal fluid, saliva, nasal swabs, etc. PCR is able to detect even 10–30 fg of M. leprae component which is equivalent to 2.8–8.3 bacilli. Several stretches of M. leprae genome are specific for ML and therefore ML-specific PCRs were developed using genes like RLEP, hsp65, 18kDa, 36kDa, 16SrRNA, sodA.,, Most of these genes have been used singly for diagnosis of leprosy. A quantitative PCR (qPCR) was used in clinical samples using RLEP,,,16SrRNA,,Ag85B,18kDa, 36kDa,,,, gene targets and it was noted that RLEP-PCR was most sensitive out of all these specified gene-based PCRs.RLEP- PCR was also used by several other workers and was found to be most sensitive and specific of all the other gene targets.,
Status of M. leprae specific PCR with treatment
ML-specific PCR can also be used for determination of the outcome after chemotherapy. As early as in 1993, a method employing–ML PCR was developed and it was noted that after 3, 6, 12, and 24 months of chemotherapy although there was no significant change in BI, the number of genomes detected by PCR reduced sharply which correlated with the reduction in the morphological index of the bacilli. In the recent past, a quantitative real-time (RT)-PCR based on hsp18 mRNA, demonstrated that after 2 years of MDT treatment, no viable ML could be detected in 47 leprosy cases; however, considerable amount of DNA could be detected in many of these samples suggesting that RT-PCR could be used effectively in monitoring patients under chemotherapy. The method using RT-PCR was not further developed because of its complexity to perform under field conditions. However, with the emergence of drug-resistant ML, this technique is presently being employed in reference laboratories in samples collected from the remote areas., Specificity and sensitivity of the PCR-based assays have been listed in [Table 1].
Use of molecular-based technology for drug resistance in leprosy
Emergence of drug resistance in leprosy has been recently reported from several countries including India., As mouse foot pad technique for the detection of drug resistance takes a minimum of 6 months, molecular-based techniques in finding mutation in drug-resistant determining region (DRDR) of ML are being used in patients who are not responding to MDT. Many of the mutations responsible for drug resistance in folP1 r egion for DDS, GyrA region for Oflaxacin, and rpoB region for Rifampicin have been shown to be responsible for resistance in ML. Therefore, slit skin smears or biopsies preserved in 70% ethanol from patients not responding to treatment could be sent to reference laboratories for finding out mutations by gene sequencing in respective DRDR regions of drugs responsible for drug resistance.
| M. Leprae -Specific Antibody or Pcr Positivity in the Context of Normal Household Contacts and Endemic Population|| |
From the discussion above, it may be concluded that he above mentioned ML-specific antibody and PCR tests are valuable tools in the diagnosis of a doubtful or a definite case of leprosy. However, clear guidelines in case of positive results of any of these assays in household contacts or an individual from endemic population are lacking. It is known that many normal household contacts of cases turn out to be leprosy cases in future, and household contacts of MB cases have been shown to have 3.8–10-fold more chance of getting leprosy than the general population.,, Several studies in Indonesia, India, and Brazil have indicated that in an endemic community as population are exposed to infection, the biological samples such as blood, nasal swabs, saliva, and slit-skin smears of contacts of patients remain positive either for ML-specific antibody or for specific component of ML.,,,,, How many of these biomarker-positive contacts of the population will transform into cases is generally uncertain and depends on the immune status of the individual having subclinical infection. These diagnostic tests are performed only once in individuals who pass through a dynamic state of the immune system, and therefore every individual who test positive to these assays do not develop leprosy in future. Rather, it has been noted in a 2-year follow-up study that large number of cases appear from the ML-specific test negative group from the community which outnumbers the cases that appear from the small cohort population of household contact group. Therefore, these tests performed only at a single point of time may not be useful for prediction of a future case. However, these tests could be applied in a cohort population at risk under surveillance but will not prove to be a cost-effective proposition for the leprosy control program.
| Conclusion|| |
The above discussion has briefly described the recent progresses that has been made in the area of specific diagnostic tests for leprosy. Despite the attempts to develop a definitive early diagnostic test for leprosy especially for patients in whom cardinal signs of leprosy are not fulfilled, the objective of an ideal diagnostic test is still to be attained. Rather, these assays fail to detect almost 60% cases of PB leprosy patients demonstrating one of the cardinal signs. Another major concern with these tests is the positive results in significant number of contacts not showing any clinical signs of leprosy. These contacts have been found to have the same level of antibodies or markers of ML in their biological samples like early cases of PB leprosy. However, these antibody-based assays using any of the antigens like PGL-1 or LID-1 or NDO-LID may prove to be useful in cases of early diffuse lepromatous or MB leprosy having no major nerve deficit or thickening which may be missed by leprosy experts. In spite of the above advancement in technology, there is still a need for development in early diagnosis of leprosy. Future efforts could be directed to search for new and novel antigens or host biomarkers which will be mainly expressed only in subclinical, preclinical, and in early leprosy cases and at the same time will also be able to discriminate these cases from uninfected endemic contacts.
The author acknowledges the help provided by Dr. Ravindra P. Turankar, Dr. Mallika Lavania, and Dr. Itu Singh for scrutinizing the manuscript. Authors also acknowledge the Leprosy Mission Trust India for providing opportunity to write this review.
A brief research background of Dr. Utpal Sengupta
Dr. Utpal Sengupta completed his B.V. Sc. in 1963 from Bengal Veterinary College, Calcutta University. Later, in 1966, he obtained M.V. Sc. (Pathology) from Indian Veterinary Research Institute, Izatnagar. Further, in 1973, he did PhD (Pathology) from Post Graduate Institute of Medical Education and Research, Chandigarh. He then specialized in Imuunopathology, Immunology, and Molecular Biological aspects and remained engaged in leprosy research from 1974 till today. He worked as a postdoctoral fellow under Dr. R.J.W. Rees at the National Institute for Medical Research, Mill Hill, London, UK. Later in 1976, he joined at the National JALMA Institute of Leprosy and Other Mycobacterial Diseases (NJIL and OMD) as Sr. Research Officer under ICMR and retired as Director of the Institute in 2001. After retirement, he worked as an Emeritus Scientist of ICMR for 5 years at NJIL and OMD. Following this, he worked as Laboratory Manager in a Bill Gates Funded project at ICMR for HIV prevalence in long distant truck drivers of Indian National Highways from 2006 to 2011. Later, in 2011, he joined as a Consultant to the Leprosy Mission Trust India (TLMTI) for overseeing the laboratory research activities of the Stanley Browne Laboratory of TLMTI. During his tenure of research in leprosy, he had published 215 research papers in leprosy journals (International Journal of Leprosy, Indian Journal of Leprosy, Leprosy Review) and in other journals with high impact factors like Nature Genetics, Journal of Infectious Diseases, Journal of Clinical Microbiology, Clinical and Experimental Immunology, Transactions of The Royal Society of Tropical Medicine and Hygiene, Microbial Pathogens, Immunology Letters, Immunology Today, Infection and Immunity, American Journal of Infectious Diseases, CMI, Human Pathogen, AIDS, Microbes and Infection, Frontiers in Immunol, etc.
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Conflicts of interest
There are no conflicts of interest.
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