Replace, Resistance, Reinfection, Reactivation in Leprosy

This presentation is for resident doctors who are pursuing their MD degree. it will help during writing exams and better understanding of the topic.

1. Replace, Resistance, Reinfection, Reactivation in Leprosy Dr. Niraj Dhinoja

2. Relapse- Usually relapse indicates a failure to treat the infection thoroughly, particularly in chronic diseases. In leprosy termination of treatment is based on the completion of recommended duration of treatment, rather than the disappearance of clinical signs and symptoms. Inadequate and incomplete treatment is the most important risk factor for reactivation and relapse. T he principal mode of assessing the efficacy of therapeutic regimens in leprosy is the “relapse rate”. A very low relapse rate over an adequate period of observation indicates that the regimen used has been effective.

3. Various definitions for relapse in leprosy: 1. Guide to Leprosy Control (WHO 1988) A patient who successfully completes an adequate course of MDT, but who subsequently develops new signs and symptoms of the disease, either during the surveillance period (2 years for PB and 5 years for MB leprosy) or thereafter. 2. Becx-Bleumink Lists Several Criteria for Relapse • New skin lesions • New activity in previously existing skin lesions • Bacteriological index (BI) 2+ or more in two sets of skin smears • New nerve function loss • Histological evidence of relapse in skin or nerve biopsy • Lepromatous activity in the eye(s).

4. 3. Relapse in Paucibacillary Patients • Boerrigter et al. “Appearance of a new skin lesion or increase in size of pre-existing skin lesion, provided there is either strong clinical or definite histopathological evidence (or both) of leprosy in such a lesion”. • Pandian et al. proposed seven criteria for defining relapse in PB: extension of the lesion (2) infiltration, (3) Erythema (4) occurrence of fresh lesions (5) Pain and tenderness of nerve (6) new paralysis of muscles, (7) bacteriological positivity • Linder et al. defined relapse, as a case which has already received a course of specific antileprosy treatment and returns with signs of active disease which require a second course of treatment. Here a course of treatment is defined as 1 month less than the actual proposed minimum duration of MDT e.g. 11 months for MB and 5 months of treatment for PB cases.

5. Relapse rate - International Federation of Anti-Leprosy Associations (ILEP) suggests evaluating the effectivity of a control program by calculating -- The percentage of the number of patients treated for relapse versus the number of those treated for the first time in the same year. If this exceeds 5%, the control program should be reassessed. There are wide variations in the estimates of relapse rates in different regions, probably due to - variations in the definition of relapse proportions of previously dapsone treated and untreated patients range of skin smear positivity in MB cases differing durations of follow-up.

6. PREDISPOSING FACTORS FOR RELAPSE- Persisters Persisting organisms or “ persisters ” consist of permanently or partially dormant organisms that have the capacity to survive in the host despite adequate chemotherapy. They have been identified in immunologically favorable sites such as Dermal nerves, smooth muscle, lymph nodes, iris, bone marrow and liver. These organisms, which are responsible for relapse, are present in about 10% of MB patients, and their proportion may be higher in cases with higher BI. Inadequate Therapy This is usually the result of clinical miscategorization of MB leprosy with few skin lesions as PB cases, who receive 6 months of MDT instead of 12 months, initially respond to treatment, and eventually relapse.

7. Irregular Therapy Irregularity in ingesting self-administered clofazimine and dapsone either due to irregular supply of drugs or noncompliance on the part of the patient will effectively result in a scenario of rifampicin monotherapy. This will lead to rifampicin resistance, and subsequent relapse. Adherence with MDT is important to minimize the risk of relapse and avoid the emergence of drug resistance. Monotherapy The relapse rate is high among patients who have received dapsone monotherapy. This is also due to the development and multiplication of resistant organisms.

8. Drug Resistance This can be- P rimary (infection with drug-resistant strains) S econdary (resistance develops as a consequence of irregular or monotherapy) It is an independent risk factor for relapse. Clustering of rpo B and fol P1 mutation in this particular population was as high as 55%, indicating transmission of resistant strains among relapse cases and contacts. High Initial BI Patients who have a high BI initially, are at greater risk of relapse after fixed duration MDT compared to patients who are smear negative or have a low BI at registration. LL patients are four times more likely to relapse than those in the other spectrum. These patients have a poor CMI, and hence, treatment cannot be stopped after 1 or 2 years.

9. Number of Skin Lesions and Nerves The number and extent of lesions including nerve lesions, when multiple, i.e. more than five and covering three or more areas of the body correlate with higher relapse rate. Mycobacterial antibodies have been found in TT leprosy with a large number of lesions and in BT leprosy with more than 10 lesions. Since this is evidence of a fairly large number of organisms, these patients may not be truly PB, and treatment with two drugs for 6 months might be considered inadequate for these patients. Lepromin Negativity Borderline patients with a positive lepromin test have been observed to have a lower relapse rate than those with a negative response.

10. Antireaction Treatment patients who had taken antireaction treatment were five times more likely to relapse than those who had not. Persistence of bacilli was assumed to be the common factor that could explain both reactions and relapse. In one study, nearly 50% of the relapsed patients had experienced previous recurrent erythema nodosum leprosum (ENL). Physiological Conditions For example, pregnancy.

11. CLASSIFICATION OF RELAPSE Kar et al. have classified relapse as follows: Early relapse (0–3 years): Because of original misclassification resulting in inadequate/insufficient duration of chemotherapy or irregular treatment. 2. Late relapse (3–10 years): Because of drug resistance, M. leprae persisters or reinfection.

12. RELAPSE INTERVAL Relapse interval is otherwise known as incubation period of relapse. It is different with monotherapy and MDT. Dapsone Monotherapy Majority (55.0–57.0%) of relapses occurred within: • 3 years in non lepromatous • 5 years in borderline • 6 years in lepromatous Multidrug Therapy • PB : Same as with monotherapy • MB : 9 years (median) The implication of these figures is that PB patients should be under surveillance for at least 3 years and MB patients for 9 years, so that majority of the relapses can be detected.

13. PB MB Age Younger age group Older age group Skin lesion Previously subsided skin lesions show signs of renewed activity, such as infiltration, erythema, increase in extent, and appearance of satellite lesions. Often, there is an increase in the number of lesions as well. Localized areas of infiltration over various parts of the body. Soft, pink, and shiny papules and subcutaneous nodules may be found at these sites, with or without a background of infiltration. Papules may enlarge to form plaques. They feel like peas in a pod, and increase in size with time. Nerve involvement New nerves may become thickened and tender, accompanied by extension of area of sensory loss and insidious onset of motor deficit. Patients may complain of aches and pains along the peripheral nerves with or without evidence of nerve damage. Relapse may occur only in nerves without skin involvement (neural relapse) Nodular swellings may occur along the course of cutaneous nerves and peripheral nerve trunks, in addition to fresh nerve thickening and/or tenderness, with insidious loss of function. Increase in area of sensory loss is noticed.

14. PB MB GENDER Male more than Female Male more than Female OTHER NAD Ocular Lesions Cases with pre-existing eye involvement may relapse with iris pearls or rarely, lepromata. Mucosal Lesions Papular or nodular lesions may be seen on the hard palate, inner lips and glans penis.

15. Tests for diagnosing relapse - Smear positivity- Reappearance of positivity for AFB after the case has become negative has been considered a feature of relapse in both PB and MB cases. T he criteria for diagnosing relapse in MB leprosy- BI persisting at the same level A n increase in BI of 2+ A ppearance of new active lesions with high BI BI becoming greater than what it originally was in the pre-existing lesion However, an increase in BI of even 1+ (checked repeatedly) should be considered as adequate supporting evidence for diagnosing relapse in patients who had earlier become negative, or were showing downward trend in BI after MDT.

16. In Vivo Techniques Mouse Foot-pad Studies This method measures the viability and includes cultivation of M. leprae. In Vitro Techniques for Assessing the Viability • Morphological index • Fluorescein diacetate-ethidium bromide (FDA-EB) staining • Laser microprobe mass analysis (LAMMA) • Adenosine triphosphate (ATP) measurements • Macrophage-based assays. Molecular Techniques • Deoxyribonucleic acid and ribonucleic acid targeting probes • Polymerase chain reaction (PCR) for gene amplification

17. IMMUNOLOGICAL TESTS FOR RELAPSE- Phenolic Glycolipid 1 Antibodies Lepromatous leprosy (LL) patients show a significant rise in titer of phenolic glycolipid 1 (PGL-1) immunoglobulin M (IgM) antibodies during the time of relapse. The dipstick assay for detection of anti-PGL-1 antibodies has been used as a simple tool for classification of patients and for identification of those patients who have an increased risk of relapse. The Natural Disaccharide-Octyl-Bovine Serum Albumin ELISA U seful test both for screening for early infection with M. leprae and for predicting a relapse, particularly in cured MB patients.

18. Interleukins-based Assays The Th1 and Th2 types of interleukin profile may be a useful method of identifying the type of relapsed leprosy. W hen BL/LL patients relapse as TT/BT type- An upgradation of cell-mediated immunity (CMI), in the form of a Th1 type of immune response R ise in levels of interferon gamma (IFN- γ ), interleukin 2 (IL-2) and immunoglobulin G2 (IgG2) antibodies positive lepromin test.

19. W hen TT/ BT patients relapse to BL/LL types- Th2 type of immune response is initiated R ise in IL-4, IL-5, IL-6, IL-10 and IgG1 production fall in the levels of IL-2 and IFN- γ Lepromin negativity.

20. HISTOPATHOLOGY- Regular skin biopsies and skin smears, at least once in 6 months, from representative lesions should be studied during the period of treatment and the following 5 years after achieving negativity. Histopathology of Relapsed Lesions in MB Leprosy In the early lesion S mall and large foci of newly arrived spindle-shaped macrophages with pink granular cytoplasm are identified along with a few small clumps of persisting foamy macrophages. Solid staining AFBs reappear in skin smears and biopsy specimens, in patients who may or may not have become completely smear-negative. W ell-established Lesion T he foamy change becomes obscured by collections of spindle-shaped and immature macrophages. Skin adnexa are markedly atrophic and scanty, and dermal nerve bundles are few and show perineurial thickening and fibrosis. Macrophages, Schwann cells and endothelial cells are packed with solid staining AFBs. Occasionally, there is infiltration by polymorphs,

21. Histopathology of Relapsed Lesions in PB Leprosy Dermal collagen is destroyed during the inflammatory process, leading to an atrophied and wrinkled appearance of healed skin lesions. Nerves undergo perineural and intraneural fibrosis. M. Lepra get buried alive in these nerves and also in the arrector pili muscle cells, thereby serving as a possible focus for relapse. The difficulty that arises in PB cases is the differentiation of relapse from type 1 reaction- Features that suggest a reaction include edema around the granuloma, dilated lymphatics and proliferating fibroblasts throughout the dermis G ranuloma usually begins as a small focus of lymphocytes and epithelioid cells, which often starts in the fibrosed nerve bundles or arrector pili muscle cells. Once the granuloma becomes well-established, it grows and involves large portions of the dermis. Therefore, in PB patients, regular 6-monthly biopsies showing disappearance of the granuloma will confirm “cure”, and reappearance of the granuloma will identify “relapse”.

22. The diagnostic criteria for relapse are: [WHO] 1. Clinical Criteria • Increase in size and extent of existing lesion(s) • Appearance of new lesion(s) • Erythema and infiltration in lesions that had completely subsided • Nerve involvement (thickening or tenderness) 2. Bacteriological Criteria Assuming a 0.5–1 log-unit/year fall in BI in a MB patient, the expected BI at the end of treatment can be calculated by E xpected BI = initial BI - years after RFT I n patients with a positive BI, if BI increases by 2+ over previous smears at any two sites and continues to be so at two examinations, it is diagnosed as relapse, provided the patient has ingested 75% of the drugs. Positivity (in a smear negative patient) at any site in skin smears for AFB at two examinations during the period of surveillance is diagnostic of relapse. Limitations of this criterion are: 1. Relapses presenting with a BI of < 2+ will not be diagnosed. 2. The quality of the slit skin smears is “the weakest link in most leprosy control programs.”

23. 3. Therapeutic Criteria This is useful when RR is suspected. The patient may be treated with prednisolone after which RR should subside completely in 2 months. If symptoms do not subside or only partially subside or lesions persist or increase under the cover of steroids, then relapse should be suspected. 4. Histopathological Criteria This includes the reappearance of already regressed/ regressing granuloma in PB cases; and increased macrophage infiltration with solid staining bacilli and increasing BI in MB cases. 5. Serological Criteria In LL cases, the measurement of PGL-1 IgM antibodies is a good indicator of relapse. The first three criteria are sufficient to make a diagnosis of relapse; criteria 4 and 5 are additional, and may be used wherever facilities are available.

24. Linder scoring system – Higher sensitivity than WHO criteria

25. Drug Resistance

26. Antileprosy Drugs and Resistance Dapsone Dapsone targets folic acid biosynthesis pathway and its effect is bacteriostatic. Dapsone is an analogue of p- aminobezoic acid (PABA) and acts as a competitive inhibitor of PABA. Dapsone binds to dihydropteroate synthase (DHPS), which is encoded by the folP1 gene and inhibits folic acid synthesis. Dapsone does not bind to DHPS by amino acid substitution in DHPS and bacilli acquire resistance to dapsone. Missense mutations at codon 53 (ACC) or 55 (CCC), coding threonine or proline, in folP1 gene confer dapsone resistance. The number of dapsone resistant cases increased after dapsone monotherapy during the 1960s and 1970s including primary dapsone resistance. Primary dapsone resistance means a resistant strain from a patient without previous administration of any anti-leprosy drugs.

27. Rifampicin - Rifampicin targets the beta ( β ) subunit of RNA polymerase, which is encoded by the rpoB gene. Rifampicin binds to the β subunit and inhibits DNA dependent mRNA transcription. R ifamipicin resistance by harboring missense mutations at codon 407, 410, 420, 425 and 427. These wi ll hinder rifampicin binding to the β subunit. Missense mutation Ser (TCC) to Lue (TCG) at codon 425 is most frequently observed. The first rifampicin resistant case was reported in 1976, in an isolate from a relapsed patient on rifampicin monotherapy, who was previously treated with dapsone.

28. Ofloxacin - Ofloxacin binds to the A subunit of DNA gyrase and inhibits DNA replication. Association between mutations within the highly conserved region of gyrA subunit coded by the gyrA gene and quinolone resistance was revealed in most resistant strains of mycobacteria. R esistantance to ofloxacin harbored the mutation Ala-Val (GCA-GTA) at codon 91 in the gyrA gene. One isolate had the mutation Gly-Cys (GGA-TGC) at codon 89. Mutations at codons 89, 92, and 95 in gyrA of M. leprae also confer quinolone resistance. In vitro study indicated association between missense mutation at 464, 502, 504 in gyrB and quinolone resistance. Prevalence of ofloxacin resistance is still low in some areas with high leprosy prevalence. Of 108 strains tested, two strains from India showed mutation in gyrA

29. Clofazimine - Clofazimine (B663, Lamprene ) is bactericidal for M. leprae. The mechanism of action has not been fully elucidated. However, one of possible bactericidal mechanism is binding to GC rich domains. Clofazimine was first used for leprosy treatment in 1962. I t was anti-inflammatory and is also used to control the type 2 reaction. Only one case in an article shows clear clofazimine resistance by mouse foot pad testing. Molecular background for drug resistance to clofazimine is still unclear due to the involvement of several different mechanisms.

30. Minocycline - Minocycline is the only tetracycline group active against M. leprae . Efficacy of minocycline against M. leprae was confirmed in 1987. It is bactericidal and its activity is additive when combined with other anti-leprosy drugs. Minocycline is used as second line anti leprosy drug according to the current MDT strategy by WHO. Tetracyclines inhibit protein synthesis by binding to the 30S ribosomal subunit, blocking the binding of aminoacyl transfer RNA to the messenger RNA ribosomal complex. To date, no minocycline resistant cases are known for M. leprae .

31. METHOD FOR SUSCEPTIBILITY ASSAY Mouse Footpad Method (MFP Method) M. leprae is still not cultivable on axenic media in vitro and susceptibility testing had been hampered, until the development of the mouse footpad assay. The first dapsone resistant case was confirmed with this method in 1964. F ootpad testing is still the gold standard for drug susceptibility in leprosy.

32. LIMITATIONS OF MFP METHOD – Cumbersome and time-consuming I s not applicable to many strains N ot suitable to obtain comprehensive data to monitor global level of resistance D oes not meet the needs of clinicians The method requires viable bacilli and chilled samples are to be sent to the laboratory within a week, which is usually far from the field.

33. Mutation Detection by Sequencing Clinical samples such as bacilli by slit skin method or biopsy are kept in 70% ethanol in room temperature. These samples are processed for DNA extraction. DRDRs are amplified by PCR and obtained PCR products are sequenced. Isolates with known missense mutation confirmed to confer drug resistance are regarded as drug resistant. DNA sequencing of PCR products is independent of the viability of M. leprae . Mutation Detection by DNA Microarray I mplementation of sequencing is not easy in many developing countries where the prevalence of leprosy is still high. To overcome this disadvantage, some simple methods, which can be carried out without any special equipment such as sequencer. S eries of oligonucleotide probes corresponding to each mutation detected in the folP1, rpoB , and gyrA genes were selected and fixed on a glass slide as capture probes. Biotin labeled PCR products were simultaneously amplified in a tube, denatured and hybridized with a probe corresponding to each mutation.

34. Buddemeyer Method Buddemeyer method is based on detection of 14-CO2 generation from 14-C- labeled palmitic acid in 7H12 medium with or without the metabolism by M. leprae. The method requires large quantity, 107 of the bacilli, for one assay. The method is not applicable for susceptibility test of clinical material, but useful for screening efficacy of a compound, for leprosy treatment. Other methods – GenoType LepraeDR R eal-time PCR

35. Reinfection

36. REINFECTION- If the person continued to live in an endemic area after RFT, he could be reinfected at any time by exogenous bacteria discharged by another untreated case. In this situation, he could eventually (after the usual incubation period) present with overt manifestations of an active infection. This would be impossible to differentiate clinically from an endogenous relapse. Relapse due to persisters is thought to be far more common that relapse due to reinfection. Occasionally, it may be possible to distinguish an endogenous relapse from a new infection by different bacteria, using molecular biology techniques, but only if samples from the time when the person was first diagnosed with leprosy (for example if the case was a subject in a research study with long follow-up) are available for comparison.

37. The bacteria present at the time of relapse may show differences as judged by genomic analysis It is a possibility when the time interval after RFT and occurrence of new lesions is beyond sufficient period of duration concordant with the incubation period of the disease, on relapse. Even after completing treatment, a lepromatous case does not become truly immune competent, and hence, although the risk of reinfection is not high, there is a definite risk. When reinfection does occur, the incubation period is bizarre and fresh skin and nerve lesions do not correspond to the original lesions.

38. Reactivation

39. Reactivation- Reactivation of lesions occurs due to incomplete treatment, i.e. premature termination of treatment or gross irregularity in treatment either due to noncompliance or irregular supply of drugs. Reactivation occurs soon after subsidence of the disease while relapses occur after complete and sustained subsidence of the disease. Much more common than a true relapse is “Reactivated disease” in cases who never completed a full course of adequate chemotherapy. However, premature cessation of therapy would leave some viable bacteria which can immediately recommence replication. After an interval, the bacteria again reach a level at which increasing clinical signs of infection re-occur, and the BI will slowly increase again.

40. The patient may have initially responded clinically to the therapy, but after a long interval (during which the drug-resistant bacteria multiply to a critical level) he deteriorates again, developing new active signs of infection. The commonest reason for re-activation of disease is non-compliance which does not only refer to “defaulting” (when the patient fails to collect his medication from clinic) but also non-consumption of medicines at home.

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