3. Serology
1. Anti Phenolic glycolipid-1 (PGL-1)
a) IgM ELISA
b) Dipstick assay using ND-O-BSA-based ELISA
c) ML-flow test
2. 35-kD based serology
3. Anti MM2 IgG
4. Anti LID-1 and NDO-LID rapid test
4. 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.
Initially ELISA based method was developed
Sensitivity- 90-95% in LL/BL vs. 0-40% in PB cases
Trisaccharide and disaccharide components of PGL-1- react specifically with IgM antibodies in patients’
sera..
Hence, 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.
1) PGL-1 anti IgM
5. 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.
Using this neoglycoconjugate, newer assays known as M. leprae dipstick assay , Particle
agglutination assay and ML-flow test were developed.
Sensitivity of ND-O-BSA dipstick assay – 94.9%
6. In order to make the ML-dipstick assay suitable for field conditions, the
neoglycoconjugate-based assay was modified by developing 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
patient serum.
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.
Sensitivity – 97.4% (MB) 40% (PB) specificity – 90.2%
7. ADVANTAGES-
1. Levels of PGL-1 antibody will decline after adequate chemotherapy. Hence useful method
to monitor leprosy patients under treatment.
2. Useful in determining the efficacy of MDT
3. Relapse – levels of antibody increases
4. Resistance – Levels of antibody remains persistent
DRAWBACK –
False positive : in highly endemic area , 10% uninfected individual may come positive.
8. 35-kD protein is present in the cell membrane of M. leprae
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)
Sensitivity – 98.5% (MB) 46.7% (PB) Specificity – 97.5%
Cross reacts with M. avium, Mycobacterium kansasii, Mycobacterium paratuberculosis, M.
smegmatis
2) 35-kD-based serology
9. The number of anesthetic patches in patient positively correlates with the level of antibody
Antibody levels were also found to correlate positively with the number of nerves involved in
primary neuritic leprosy
Antibody level against 35kD was found to decline following effective chemotherapy of patients.
10. Considering a low level of false positivity with PGL-1 antigen, Antigens which reacted strongly
with patients’ sera and minimally with control sera, were selected for further analysis.
The proteins selected were ML0405 and ML2331. These two proteins have been made as a
fusion construct and have been named as LID-1 [Leprosy Infectious Disease Research Institute
Diagnostic-1]
A rapid test based on NDO-LID has been developed and has been named as NDO-LID rapid test
for field purpose/point-of-care.
3) Anti LID-1 and Anti NDO-LID antibody
11. Serum sample (10 µl) and running buffer (100 µl) are charged in the well. The reaction of the
test and control yields a red color. Readings are recorded within 20 min of charging of samples.
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.
Anti NDO-LID Sensitivity – 95% (MB), 75%(PB) and specificity – 96.1%
Advantage- positivity seen one year before development of clinical MB and detection of anti
PGL-1 antibody.
Antibody level to LID-1 decline more rapidly after MDT regimen compared to that of
PGL-1-antibody level
12. Cytokine profile in leprosy
Leprosy develops in a patient mainly due to altered host response to M. leprae which depends
on microbiological and immunological characters of the individuals.
Studies on cytokines revealed involvement
TT leprosy - Th1 cytokines like interleukin-2 and IFN-γ
LL patient - Th2 type cytokine like IL-4, IL-5 and IL-10
14. Polymerase chain reaction (PCR)
•First done by Williams in 1990.
•To detect M. leprae DNA by extraction , amplification and identification.
•Specimens : skin biopsy, skin sections/paraffin blocks, skin smears, nerve sections, biological fluids (blood,
pleural effusion, ascetic fluid, CSF, saliva, nasal swab) etc.
•PCR is able to detect even 10-30fg of M. leprae (equivalent to 2.8-8.3 bacilli)
•ML specific gene sequences used – RLEP, hsp65, 18kDa, 36kDa, 16SrRNA, sodA
•RLEP-PCR is most sensitive and specific of all other gene targets
•To know viable bacilli –
15. Types of PCR
Reverse Transcriptase PCR – since PCR is
based on DNA detection , did not reflect
live bacilli
Real Time Quantitative PCR : measure
total DNA content
Correlate with BI (molecular BI)
So RT –PCR developed which is basically
RNA detection- reflect nucleic acid from
only live organism
Here 16SrRNA gene used. Biopsy sample
is sent in RNALater
16. 1. In case of diagnostic dilemma
2. Pure Neuritic Leprosy – inconclusive biopsy report
3. To differentiate between relapse and reaction by detecting viable bacilli.
4. To differentiate reinfection from relapse
5. Suspected drug resistance
6. To detect early disease in household contact
7. In monitoring patient under chemotherapy
ADVANTAGES
17. q(RT)-PCR or Real-Time PCR based on hsp18mRNA, shows no viable ML detected after 2years of
MDT, however considerable amount of DNA could be detected in many samples suggesting that
RT-PCR could be used effectively in monitoring patients under chemotherapy.
Status of PCR after treatment ?
18. Mouse foot pad technique for detection of drug resistance takes minimum 6 months.
PCR used to find the mutation in drug-resistant determining region (DRDR) of ML by gene
sequencing, i.e.,
1. folP1 region for dapsone
2. GyrA region for ofloxacin
3. rpoB region for rifampicin
Specimen - SSS or Biopsy preserved in 70% ethanol
For mouse foot pad & PCR, fresh biopsy specimen (BI 2+) to be sent and to be inoculated in mouse
foot pad with in 2 hr.
PCR for drug resistance !
19. PCR using RLEP: specificity 100%; Sensitivity 73.6% (In PB- 73%; MB- 100%)
Can detect 53% of BI negative leprosy cases
PCR using 16SrRNA : specificity 50%; Sensitivity 100% (PB- 51%; MB- 100%)
Duplex-droplet digital PCR – more accurate than Qpcr in quantifying (give accurate number of
initial template nucleic acid)
- greater sensitivity
- can detect gene expression variation <30%
20.
21.
22. High Resolution Ultrasound in leprosy
HRUS is a non-invasive, imaging technique, which provides real time examination of deeper
tissues including peripheral nerves in static and dynamic states such as blood flow.
Since the hall marks of leprosy are nerve enlargement and inflammation, HRUS and colour
Doppler imaging can be used to demonstrate nerve damage.
Peripheral nerve ultrasound provides information on the exact location of nerve enlargement
and morphological alterations in the nerve including echo texture, fascicular pattern and
vascularity.
More useful in pure neuritic leprosy
23. Colour Doppler imaging - Normally there is hypo-vascularity of nerve trunks. Increased blood
flow signals seen in colour Doppler in thick and tender peripheral nerves of leprosy denotes
oedematous and hyperaemic changes secondary to inflammation leading to alteration of an
effective blood nerve barrier during reactions.
Other imaging studies- MRI (Magnetic Resonance Imaging) and nuclear magnetic resonance
25. Why newer / alternative treatment regimen?
• Current WHO-MDT regimen is too long
• Compliance issues – patient who cannot take Clofazimine and Rifampicin need alternative
regimen
•Daily dose of Dapsone and clofazimine cannot be supervised
•To help in drug resistant cases
•To develop a common regimen for PB and MB leprosy
26. Ideal characteristic of a new regimen
•It should be oral
•Should be bactericidal and should not antagonise rifampicin in combination
•Should not have debilitating side effect
•Should be first tested in labs
•Bactericidal effects should be studied in clinical trials
•Long term relapse rate should be assessed
29. RIFAPENTIN (P)
Derivative of rifampicin with
Peak serum concentration
Serum T1/2 > Rifampicin
More effective
MOXIFLOXACIN (Mx)– More active and bactericidal than ofloxacin
Bactericidal activity identical to rifampicin
P > R = Mx > O or Clarithro or M > Clofa or Dapsone
30. Alternative regimens
ROM (Rifampicin 600 + Ofloxacin 400 +
Minocycline 200mg )
(Single dose or multiple dose monthly
for 12months)
• Studies shows single dose in PB ineffective
• Multiple doses showed equal efficacy that of WHO-MDT in
MB patient but with high rate of relapse. So multiple dose
can be considered in PB
• Advantage – fewer side effects
monthly supervised dose
Rifampicin + Ofloxacin for 28 days Far more risk of relapse
Uniform MDT (Rif+Clof+Dap) MDT for
6months for PB and MB)
• Appears promising - effective and well tolerated in patients
with PB leprosy
• Adv – MB cases misclassified as PB can be adequately treated
Concerns :-
• Seems to be too short in MB leprosy (more reactions than
those on MDT in some studies)
• Regarding relapse long term followup more than 7 yrs needed
before concluding
• Addition of clafazimine in PB will add to side effects may
31. Moxifloxacin based regimen
Rifampicin sensitive
(PMMx)
Rifapentin 900mg (or Rifampicin 600mg)
+
Moxifloxacin 400mg
+
Minocycline 200mg (or Clarithromycin 1g)
Rifampicin resistant INTENSIVE PHASE – daily for 6months
Moxifloxacin 400mg
+
Minocycline 100mg
+
Clarithromycin 500mg
+
Clofazimine 50mg
2009 suggestion by “WHO report of the global programme managers” meeting on “Leprosy Control Strategy”
Monthly supervised
for 12months
CONTINUATION PHASE- monthly for 18months
Moxifloxacin 400mg
+
Clarithromycin 1000mg
+
Minocycline 200mg
32. Other newer drugs
Gatifloxacin : flouroquinilone antibiotic
Dose – 100-400mg/day
Linezolid : Dose 600mg per day
can cause maroow suppression or optic atrophy
33. Limitations of newer drugs
1. PB patient treated with ROM therapy were twice as likely to have a relapse
2. There are insufficient data to conclude on the efficacy of multidose ROM therapy in MB
leprosy
3. Though bactericidal activity of moxifloxacin and rifapentine are comparable to rifampicin,
they are too costly to afford
4. The effectiveness and possible sideeffects of newly proposed drug regimen must be carefully
tested in controlled trials and in field trials before being applied to patients
34. Prophylaxis
Chemoprophylaxis :
◦ Previously used Dapsone or
◦ LPEP was launched globally to evaluate the feasibility and efficiency of contact tracing and
provision of preventive treatment for leprosy and to determine its impact on leprosy incidence
◦ Under this, the close contact of leprosy case were screened; symptomatic patients referred for
MDT and others were given SDR to decrease their risk for developing leprosy by 50-60%
◦ Dose : >35kg- 600mg ; 20-35kg – 450mg; <20kg- 10-15mg/kg
◦ Only few trials available on this
◦ Limitations :
◦ Chemoprophylaxis is confined to household contacts only
◦ Effect is temporary(<2yrs) and has to be repeated
◦ Implementation studies needed to assess acceptability and feasibility
◦ Potential risk of rifampicin resistance - but a review study of multidisciplinary experts
concluded that SDR given in contacts, in the absence of symptoms of active TB , poses a
negligible risk
35. Immunotherapy
• Manifestation of disease depends to a large extent on host immune response.
• Immunomodulators are required to modulate immune response which damages nerves
in host.
36. EFFECTS OF IMMUNOTHERAPY
Promotion of CD4 Th1 cells effective antibacterial process
Overproduction of CD4 Th2 cells is switched off.
Regulatory activity of CD8 cells is relaxed to allow Th1 activity
More efficient killing viable bacilli including persisters.
37. OUTCOME
Faster clearance of dead & viable bacilli including persisters leads to-
DECREASE IN
-Duration of treatment
-Morbidity & mortality
-Transmission & relapse
-Incidence & severity of
reaction
INCREASE IN
-Case holding & better
compliance
-Clinical improvement
-Improved host immunity
38. CLASSIFICATION OF IMMUNOMODULATORS-
Related mycobacteria sharing antigens with M
leprae(Vaccines)
Drugs
Miscellaneous agents or components of M.
leprae which mount an immunogenic response
39. Leprosy Vaccine
Regulate & optimize immune system –
-Rapid clinical & bacteriological improvement.
-Prevention of disease on exposure to pathogen
-Granuloma clearance.
-Treatment and decrease incidence of reactions.
-Prevention of nerve damage.
Vaccine can be used for
Prophylactic Therapeutic
40. CLASSIFICATION OF CANDIDATE VACCINES-
1st GENERATION-
NON CULTIVABLE(M leprae) CULTIVABLE M. leprae
1. Killed M leprae 1.BCG
2.Killed M leprae + BCG 2.BCG+ M. vaccae
3.Acetoacetylated M. leprae 3. Killed M. welchii
4.Killed ICRC
5.M. vaccae
6.M. habana
7.M. gordonnae
8.M. phlei
2ND GENERATION(IN VITRO/ANIMAL STUDIES ONLY)
Subunit vaccine
Shuttle plasmid vaccines
41. BCG Vaccine
• Indian studies- protective efficacy ranging from 20-70%
• Additional dose of BCG more protective in prevention than single dose where leprosy continues to
be a public health problem (At the start of MDT & every 6 months till end of treatment)
• Faster attainment of smear negativity & faster fall in BI, More rapid killing of bacilli.
• Increased risk of type 1 reaction, tuberculoid & indeterminate leprosy.
BCG + KILLED M. leprae
• The combined vaccine was tested worldwide but was not more effective than regular BCG.
42. ICRC (Indian cancer research center) Vaccine
◦ Cultivable mycobacteria belonging to M. avium intracellulare complex
◦ More accessible making the organism a stronger immunogen.
◦ From a cultivable organism & hence cheap.
◦ No contamination with animal products.
◦ Induce stable immunity
43. MiP Vaccine(Mycobacterium indicus pranii)
Earlier known as Mycobacterium w
NLEP has introduced MiP vaccine in a project made in India from the year 2016.
Shown to have both immunotherapeutic and immune-prophylactic effects in multibacillary
leprosy patients and their contacts in both hospital and population-based trials
It also reduced the bacillary load, upgraded the lesions histopathologically, led to complete
clearance of granuloma, reduced reactions and neuritis and reduced the duration of MDT in
leprosy patients.
In the new field project undertaken under ICMR and NLEP, the index leprosy patient will receive
the MIP vaccine over and above the MDT. His family members and contacts would be immunized
with MIP twice at an interval of 6 months.
