Detection of Mycobacterium leprae DNA in skin lesions of leprosy patients by PCR may be affected by amplicon size

Despite the high sensitivity and specificity of PCR for infectious disease diagnostics, it has presented low sensitivity for Mycobacterium leprae DNA detection in the tuberculoid pole (TT and BT) of leprosy. In order to demonstrate the effect of amplicon size on the efficacy of PCR detection of M. leprae DNA in skin lesions of leprosy patients, two pairs of primers targeting the M. leprae genomic DNA, RLEP3 (X17153), were used to amplify fragments of 372 and 130-bp until their PCR end-points were reached after 40 reaction cycles. Skin biopsies of leprosy lesions in 110 non-treated patients were used for bacilloscopy index (BI) analysis and PCR tests. The 130-bp fragment was detected in 73.6% of samples (81/110), and classified as TT (40%), BT (55.5%), and 100% of BB, BL and LL. The 372-bp fragment was detected in 52.7% and classified as TT (13.3%), BT (33.3%), BB (64.7%), BL (83.3%), and LL (95.2%). The BI of biopsies was positive in 39.1% of samples, classified as TT (0%), BT (2.2%), BB (64.7%), BL (91.6%), and LL (95.2%). The shorter amplicon (130-bp) has improved diagnosis by 20.9 and 34.5% in relation to the 372-bp fragment and the BI, respectively, and has shown a superior sensitivity (73.6%), specificity (100%) and accuracy (86.2%). The 130-bp amplicon could not detect % of positive BI of biopsies in BT cases. Therefore, for confirmatory diagnosis, we propose the use of PCR detection of the 130-bp genomic target, especially when the tuberculoid pole forms are considered, which has reached 51.6% of positivity in this group.     

Development of a mouse food pad model for detection of sub clinical leprosy

Early diagnosis of leprosy and a multi-drug therapy (MDT) regimen will block the trajectory of nerve damage, disability and deformity that are the hallmarks of this chronic disease. However, the diagnosis of leprosy is made solely by recognition of clinical signs and symptoms, requiring special expertise. These limitations also result in the under reporting of worldwide prevalence and incidence rates for leprosy. Sorely needed is an objective laboratory test for detecting early leprosy. As the antigenic burden of M. leprae can be virtually undetectable in early clinical leprosy, cell mediated immunity and antibody responses will likely be weak. So the sensitivity of new diagnostic tests is as important as specificity. Major efforts are underway employing recombinant M. leprae antigens and synthetic peptides, to develop diagnostic assays for early leprosy infection, using in vitro T cell reactivity or serological tests. We have used the initial phase of the mouse foot pad model as an 'early' model of leprosy infection to screen T cell responses against M. leprae specific antigens and synthetic peptides. Unlike human disease in animal models we can control infection progress and monitor bacillary growth relative to time course of development of T cell response to specific M. leprae antigens. The study employed splenic T cells instead of draining lymph node T cells to model the systemic response as opposed to a local one. We found that 10(5) live M. leprae is the minimum dose required for any meaningful and consistent in vitro splenic IFN-gamma response against M. leprae antigens 3 months after foot pad inoculation. Using this model we found that several M. leprae recombinant proteins, ML0840, ML2028, ML2307, ML2346, ML2478, and ML2532, induced significant levels of IFN-gamma secretion. By controlling for variables that can be confounding factors in the sensitivity of human testing, this mouse model provides an interface between M. leprae diagnostic antigen development and the screening of these antigens in humans under field conditions.     

S-100 as a useful auxiliary diagnostic aid in tuberculoid leprosy

BACKGROUND: The diagnosis of tuberculoid leprosy is often difficult on hematoxylin and eosin (H&E) due to the absence of demonstrable nerve destruction. This study evaluates the utility of S-100 staining in identifying nerve fragmentation and differentiation of tuberculoid leprosy from other cutaneous granulomatous diseases. METHODS: Fifty cases of leprosy including 38 borderline tuberculoid (BT), two tuberculoid (TT), and 10 indeterminate leprosy (IL) were studied. Eleven controls of non-lepromatous cutaneous granulomatous lesions were included. S-100 was used for identifying the following dermal nerve patterns: infiltrated (A), fragmented (B), absent (C), and intact (D) nerves. RESULTS: On H&E, only 18/38 (47.4%) BT cases and 1/2 (50%) TT cases revealed neural inflammation. On S-100 staining of BT cases, 28/38 (73.7%) showed pattern B followed by patterns C and A in 8/38 (21.1%) and 2/38 (5.3%) cases, respectively. Both the TT cases showed pattern B. Only intact nerves (D) were seen in all the control cases. S-100 identified nerve damage in 4/10 (40%) IL cases. The patterns A, B, and C had sensitivity, specificity, and positive and negative predictive values of 100% in diagnosing tuberculoid (BT + TT) leprosy. CONCLUSIONS: S-100 is superior to H&E in identifying nerve fragmentation (p < 0.01). It also aids the differential diagnosis of tuberculoid leprosy.     

Alpha-1-antitrypsin in leprosy

Estimation of Alpha-1-antitrypsin (AAT) levels was carried out in 52 patients of various types of leprosy. Fifty age and sex matched healthy individuals served as controls. The mean level of AAT in controls was 290.12 +/- 59.56 mg/dl. In patients of tuberculoid leprosy (TT), borderline tuberculoid leprosy (BT) and borderline leprosy (BB), the AAT levels were found to be 284 +/- 47.03, 314.37 +/- 31.56 and 324.44 +/- 32.05 mg/dl respectively. These were statistically insignificantly raised when compared with controls. In borderline lepromatous leprosy (BL), lepromatous leprosy without erythema nodosum leprosum (LL without ENL) and in LL with ENL there was a statistically significant rise in AAT levels. The maximum levels of AAT were observed in patients of LL with ENL (mean 500.8 +/- 93.44 mg/dl. P less than 0.001).     

Mycobacterium leprae reactive T cell clones from lepromatous leprosy patients after prolonged dapsone chemotherapy

The proliferative responses of peripheral blood mononuclear cells (PBMC) to Mycobacterium leprae and BCG were studied in two groups of leprosy patients: a group of 8 lepromatous patients who had been on treatment for more than 20 years (TLL) and a group of 8 untreated lepromatous leprosy patients (ULL). The mean response to M. leprae of the TLL group was 6195 cpm with 5 of the 8 patients responding positively. The mean response to M. leprae of the ULL group was 617 cpm, with only 1 patient showing a positive response. The corresponding proliferative responses to BCG were 19,908 cpm in the TLL group and 7908 in the ULL group. Thirteen M. leprae reactive clones were established from 2 TLL patients and 5 M. leprae reactive clones were established from 2 tuberculoid leprosy patients. Seven of these clones, 4 from the TLL patients and 3 from the tuberculoid (TT) patients could be studied further. Three of the TLL clones responded specifically to M. leprae, while one of the clones exhibited a broad cross-reactivity to other mycobacteria. All of these clones were of the CD4+CD8- phenotype. Our findings suggest that responsiveness to M. leprae can be detected in vitro in a proportion of LL patients who have undergone prolonged chemotherapy, and that this response involves M. leprae reactive CD8+CD8- T cells, of which some appear to be specific to M. leprae.     

Leprosy in children: correlation of clinical, histopathological, bacteriological and immunological parameters

The study of leprosy in children has indicated an incidence of 10% amongst leprosy patients attending the clinic. The duration of the disease was usually less than 2 years. The expression of leprosy in this group was either a macule and/or a plaque. Classification was, therefore, different and conforms to indeterminate (I), borderline tuberculoid (BT) and borderline (BB) leprosy. Only occasionally were other clinical variants seen. The bacteriology was largely unproductive by slit-skin smears. The lepromin (Mitsuda) responses were positive in BT and unpredictable in BB patients. Epicutaneous responses to sensitization with dinitrochlorobenzene (DNCB) paralleled responses to lepromin. Microscopic pathology was of very little help. The correlation of these parameters was only 50-60% indicating that the diagnosis of leprosy should primarily be based on clinical features.     

Evaluation of diagnostic role of in situ PCR on slit-skin smears in pediatric leprosy

A large proportion of early cases of leprosy in children remain AFB negative in skin smears. Such cases required additional techniques to confirm the diagnosis. In situ PCR on slit- skin smears is minimally invasive and less cumbersome as compared to skin biopsies. This study was initiated in our institute with the objective to evaluate the diagnostic value of in situ PCR on slit- skin smears in pediatric leprosy. A total of 25 cases of leprosy below 16 years of age were included in the study. After detailed history and thorough clinical examination, informed consent was obtained from the parents of children for slit- skin smears from lesion sites for AFB staining and for in situ PCR technique. Cases were clinically categorized according to IAL classification into indeterminate (I), tuberculoid tuberculoid (TT), borderline tuberculoid (BT), borderline borderline (BB), borderline lepromatous (BL) and lepromatous (LL). Most of the patients (76%) were between 9-16 years of age and 64% of the cases had history of contact with leprosy patients within the family. Skin smears were positive for AFB in only 20% of the cases. On applying in situ PCR, it was observed that 62.5% cases of I/TT/BT/BB category and 88.8% of BL/LL category gave positive signals. Overall in situ PCR confirmed the diagnosis in 72% cases while by slit smears diagnosis was confirmed in only 20% of cases. Further, out of 20 skin smear negative cases, 13 were positive by in situ PCR. Specificity of the signals of in situ PCR was established by demonstrating the absence of signals in nonleprosy dermatological conditions of vitiligo and P.alba. This study supports the potential usefulness of in situ PCR on slit- skin smears of early pediatric leprosy cases. This strategy will be especially useful in cases where skin smears are negative and in those cases where skin biopsy can not be done either because of unusual locations of lesions or because of sensitive age of the patients.     

Erythema nodosum leprosum: reactional leprosy

The different clinical forms of leprosy are mainly related to the variety of immunological responses to the infection. Thus, lepromatous leprosy occurs in patients with a poor cell-mediated immunity to Mycobacterium leprae, whereas tuberculoid leprosy is associated with a high resistance to leprosy bacillus. Intermediate forms, including borderline tuberculoid leprosy, borderline lepromatous leprosy, and borderline leprosy, are a continuous and unstable spectrum of the disease. Leprosy reactions are rare and not well-known states that interrupt the usual chronic course and clinical stability of patients with leprosy. They are expressions of immunological perturbations. Attending to the clinical and histopathological manifestations, leprosy reactions may be separated in 2 or 3 different variants: reverse reaction (type I), erythema nodosum leprosum (type II), erythema polymorphous (type II) and Lucio's phenomenon, mainly considered a type II reaction, but sometimes designated type III. Type I leprosy reaction, also named "upgrading reaction," occurs in borderline leprosy states and is associated with a shift toward the tuberculoid pole. Type II reaction usually occurs in lepromatous leprosy, and there are 3 different clinical variants, including erythema nudosum leprosum, erythema polymorphous-like reaction, and Lucio's phenomenon.     

Evaluation of leprosy patients with 1 to 5 skin lesions with relevance to their grouping into paucibacillary or multibacillary disease

BACKGROUND: Patients with 1 to 5 skin lesions are arbitrarily categorized as belonging to the paucibacillary (PB) group for treatment purposes. With the decreasing prevalence of leprosy in India and modifications in leprosy program, the relevance of this grouping needs further study. AIMS: To study a group of leprosy patients with 1 to 5 skin lesions and compare the clinical parameters with histopathological findings and bacteriological status of the skin and nerve to evaluate the relevance of this grouping. METHODS: Seventy seven patients of leprosy with 1 to 5 skin lesions were included in the study. The number of skin lesions was recorded. Slit skin smears (SSS) and skin biopsies were taken in all patients and nerve biopsy was performed in 19 of them. The biopsies were evaluated for the type of pathology and AFB status. RESULTS: In these 77 patients (single skin lesions, 42; two lesions, 18; three lesions, 10; four lesions, 5; and five lesions, 2 patients) the clinical classification was indeterminate leprosy (IL) in 4, tuberculoid leprosy (TT) in 4 patients and borderline tuberculoid leprosy (BT) in 69 patients. Skin smears were positive only in 1 patient. The histological diagnoses in the skin were IL in 13, TT in 3, BT in 48 and borderline lepromatous (BL) in 4 patients. Acid-fast bacilli (AFB) were found in 14 out of 77 skin biopsies. Of the 19 nerve biopsies, 17 showed histological features of BT leprosy; of these, 12 demonstrated AFB on Fite staining. The bacillary index of granuloma (BIG) ranged from 1+ to 2+. The clinico-histopathogical correlation was 63% in the BT group, with 4 patients of this group showing features of BL on histopathology. When the presence of AFB was assessed, the percentage of positivity was 1.3% in SSS, 18% in skin biopsies and 63% in nerve biopsies. CONCLUSION: Our results point to the non-homogeneous nature of this group of leprosy patients with 1 to 5 skin lesions, with varied bacteriological and histopathological features. The significance of MB type findings on histopathology in patients grouped as PB leprosy should be resolved so that these patients may be given the drug therapy and the duration of therapy they warrant.     

Diagnosing leprosy: revisiting the role of the slit-skin smear with critical analysis of the applicability of polymerase chain reaction in diagnosis

Background Diagnosing leprosy is challenging, especially in early‐stage cases, and the need for a sensitive diagnostic tool is urgent. Polymerase chain reaction (PCR) holds promise as a simple and sensitive diagnostic tool, but its usefulness in the Indian context requires further evaluation. Slit‐skin smear (SSS) remains the conventional method of leprosy detection. Hence, this study was undertaken to evaluate and compare the diagnostic efficacy of PCR versus that of SSS. Methods Punch biopsy of skin and SSS were obtained from the active margins of lesions. Cases were clinically grouped according to whether they were multibacillary (MB) or paucibacillary (PB) and classified into tuberculoid (TT), borderline tuberculoid (BT), borderline lepromatous (BL), lepromatous (LL), histoid, and indeterminate groups after clinicopathological correlation. DNA was extracted from biopsy specimens, and multiplex PCR was carried out incorporating primers intended for the amplification of a specific 372‐bp fragment of a repetitive sequence of Mycobacterium leprae DNA. Results Among 164 patients, PCR was positive in 82.3%. The sensitivity of PCR was significantly greater (P < 0.0001) than that of SSS in both the MB (85.9% vs. 59.8%) and PB (75.4% vs. 1.8%) subgroups; the difference in sensitivity in the PB subgroup is remarkable. Positivity by PCR and SSS was found in 100% of LL and histoid leprosy, but PCR had significantly greater (P < 0.0001) positivity in BT leprosy and was of definite increased value in indeterminate and TT leprosy. Conclusions Polymerase chain reaction had higher sensitivity compared with SSS, especially in diagnostically challenging and PB cases. Thus, the use of this costly but sensitive tool should be restricted to this subgroup, because SSS is sufficiently sensitive in the diagnosis of LL and histoid leprosy.     

T regulatory cells (TREG)(TCD4+CD25+FOXP3+) distribution in the different clinical forms of leprosy and reactional states

BACKGROUND

Leprosy is characterized histologically by a spectrum of different granulomatous skin lesions, reflecting patients'' immune responses to Mycobacterium leprae. Although CD4+CD25+ FoxP3+ T regulatory cells are pivotal in the immuneregulation, presence, frequency, and distribution of Tregs in leprosy, its reactional states have been investigated in few studies.

OBJECTIVES

This study aimed to verify the frequency and distribution of regulatory T cells in different clinical forms and reactional states of leprosy.

METHODS

We performed an immunohistochemical study on 96 leprosy cases [Indeterminate (I): 9 patients; tuberculoid tuberculoid: 13 patients; borderline tuberculoid: 26 patients; borderline borderline: 3 patients; borderline lepromatous: 8 patients; lepromatous lepromatous: 27 patients; reversal reaction: 8 patients; and erythema nodosum leprosum: 2 patients].

RESULTS

FoxP3-positive cells were present in 100% of the cases with an average density of 2.82% of the infiltrate. Their distribution was not related to granulomatous structures or special locations. There was a statistically significant increment of FoxP3 expression in patients with leprosy reversal reactions when compared with patients presenting with type I leprosy (P= 0.0228); borderline tuberculoid leprosy (P = 0.0351) and lepromatous leprosy (P = 0.0344).

CONCLUSIONS

These findings suggest that Tregs play a relevant role in the etiopathogenesis of leprosy, mainly in type I leprosy reaction.     

Circulating CD4+ CD25 high FoxP3+ T cells vary in different clinical forms of leprosy

Background CD4⁺ CD25^highFoxP3⁺ regulatory T cells (T‐regs) were reported to increase in chronic infections. We aimed at studying their frequency in leprosy to investigate their role during Mycobacterium leprae infection. Methods Using flow cytometry, the frequency and FoxP3 expression of circulating T‐regs was assessed in 38 leprosy patients and 38 healthy controls. Patients were divided into; group I tuberculoid (TT), group II borderline cases [borderline tuberculoid (BT), borderline (BB), and borderline lepromatous (BL)], group III lepromatous (LL), and group IV erythema nodosum leprosum (ENL). Results Mean T‐regs% and FoxP3 expression were significantly elevated in patients (particularly TT) compared to controls (3.8 ± 2.5% vs. 2.5 ± 0.8% and 78.8 ± 56.2% vs. 55.8 ± 15.7%, respectively) (P < 0.05). Comparing the four disease groups, T‐regs% was significantly different (median 5.3% in group I, 3.4% in group II, 2.8% in group III, and 1.2% in group IV; P = 0.005). FoxP3% on T‐regs was not significantly different between them [median 71.5% in TT, 62.3% in borderline categories, 67.75% in LL, and 85.75% in ENL; P = 0.149). Notably FoxP3 expression was significantly higher in ENL than controls (P = 0.011). Conclusion The frequency and suppressive marker of circulating T‐regs are elevated in TT patients. Patients with LL and ENL express significantly lower frequency of T‐regs and higher FoxP3 expression (in ENL), consistent with disease progression and immune hyper‐activation in these disease categories. Thus, rather than being detrimental to immunity, intact T‐regs activity may be beneficial to leprosy patients.     

An appraisal of enzyme linked immunosorbent assay (ELISA) and serum antibody competition test (SACT) in leprosy.

Seventy-eight untreated leprosy patients, 104 treated patients and 105 healthy contacts were tested using two serological tests, SACT (serum antibody competition test based on competitive inhibition of monoclonal antibody binding to the MY2a determinant of M. leprae) and ELISA (measurement of IgM antibodies to the neoglycoproteins D-BSA and ND-BSA representing the phenolic-glycolipid antigen of M. leprae). The controls included normal healthy individuals, patients with sputum positive pulmonary tuberculosis, and active cases of rheumatoid arthritis from the department of rheumatology. The specificity of SACT was found to be very high. ELISA was found to be positive in two patients with rheumatoid arthritis, one each for D-BSA and ND-BSA ELISA. Both tests had a high sensitivity in BL and lepromatous patients. The sensitivity to both tests was considerably lower in tuberculoid and BT patients i.e., below 40%. Therefore the diagnostic value of a negative test in suspected cases of leprosy was very low employing either of the two tests. A proportion of patients with paucibacillary tuberculoid and BT leprosy were positive after six months or longer after therapy. Similarly a large number of BL and lepromatous patients were positive after considerably longer periods of treatment. The use of either tests for determining the duration of therapy is therefore limited. SACT appears to be more sensitive than ELISA with ND-BSA in detecting subclinical infection. The cumulative positivity of the two tests may be used as a measure of the infectivity of the disease in the community and for evaluating disease control methods.     

An attempt to produce experimental tuberculoid leprosy in the nine-banded armadillo.

In an attempt to produce experimental tuberculoid leprosy, three nine-banded armadillos, two with borderline tuberculoid lepromin reaction, and one with tuberculoid lepromin reaction, were chosen. They were injected subcutaneously in a four square centimetre area in the abdominal skin with saline suspension of 6.5 x 10(7) M. leprae. Induration of skin at the injected site appeared in 24 hours and persisted for 6 months in one and for 18 months in the other two animals. Histopathological examination of the infected site at 6 weeks, 18 and 20 months showed progressively decreasing granulomatous inflammation; but the cutaneous nerves were uninvolved. Autopsy examination of the three animals failed to show disseminated disease. Since there was no evidence of nerve involvement, experimental transmission of tuberculoid leprosy to armadillos could not be established in this study.     

Differential expression of Langerhans cells in the epidermis of patients with leprosy

Eighteen patients with lepromatous leprosy (LL) showed a significant reduction (P less than 0.001) of Langerhans cells (LC) irrespective of whether the biopsies were obtained from involved (398 +/- 186) or healthy skin (304 +/- 98). The cells showed morphological changes consisting mainly of loss of dendritic processes. Twenty-four controls (age, sex and race matched) had a mean number of LC of 632 +/- 138. In tuberculoid patients (TT) significant differences were observed, depending on the site of biopsy. Nine biopsies from involved skin had 993 +/- 206 LC, whereas 11 from healthy skin had 448 +/- 96 (P less than 0.001). This difference was confirmed in six additional borderline tuberculoid (BT) and TT patients in whom biopsies were simultaneously obtained from involved (973 +/- 179) and uninvolved skin (498 +/- 99). In 10 patients with indeterminate leprosy the LC density did not differ from the control population (630 +/- 261). The expression of LC numbers in BT and TT patients may represent migration of these cells from healthy skin to involved areas or mobilization of a central pool. The low density found in LL patients could interfere with adequate presentation of mycobacterial antigens leading to tolerance. Alternatively the presence of T helper cells in TT infiltrates may produce factors that recruit LC; their absence in LL lesions may account for the decrease in LC expression.     

Utility of gelatin particle agglutination test (MLPA) for rapid serodiagnosis of leprosy in a hyperendemic area.

The anti-PGL M. leprae specific antibodies were estimated by MLPA test in 79 patients of leprosy, 8 contacts of lepromatous cases and 10 healthy controls in a hyperendemic area. The results indicated an over all seropositivity of 50.6% in leprosy patients. Three of the eight contacts and five of the controls also gave positive results. Higher seropositivity rates were noted in multibacillary patients (73% in lepromatous, 53.6% in borderline, 40% each in tuberculoid and indeterminate and 10% in pure neuritic types). The practical application of MLPA test in its present form as a serodiagnostic procedure for screening subclinical or clinical infections in leprosy patients appear to be of limited value in hyperendemic areas. Further studies involving large series of subjects are necessary for reaching definite conclusions.     

Correlation of clinical, histological and immunological features across the leprosy spectrum

The Ridley-Jopling system of classification of the variegated clinical pattern of leprosy is based on the specific cell-mediated immunity observed in the histopathology of skin lesions conforming to a spectrum from TT at one end to LL at the other. In this study a fairly large sample of 90 patients was classified on clinical grounds; the histopathology of the skin lesions was studied blind. There was an overall concordance of 90% between the clinical and histological classifications. In addition, the systemic cell-mediated and humoral immune responses were studied. The in vivo cell-mediated immune response, namely the Mitsuda skin response, mostly conformed to the clinical classification. While the in vitro lymphoproliferative responses to BCG and its sonicate were high, the lymphoproliferative responses to Dharmendra lepromin were surprisingly poor. Humoral responses to 35 kDA protein of M. leprae and PGL-1 were good in most LL, BL patients and tapered off towards TT. IgG antibodies to recombinant ML 65 kDa proteins denoted mycobacterial presence.     

Treatment of paucibacillary leprosy with a regimen containing rifampicin, dapsone and prothionamide.

Ninety paucibacillary leprosy patients having indeterminate (I), tuberculoid (TT) and borderline tuberculoid (BT) type of leprosy with bacterial index (BI) of less than two on the Ridley scale were treated with rifampicin (RFM) 600 mg once a month, dapsone (DDS) 100 mg daily and prothionamide (PTH) 250 mg daily. Treatment was stopped at the end of six months. The patients tolerated the drugs fairly well and in only two patients the drugs had to be stopped (in one due to jaundice and in the other due to gastric intolerance). About 6% of patients had early reactions which subsided with additional steroid therapy. The inactivity rate was 60% at six months and this improved to 96% at 12 months. No cases of late reactions and relapses were encountered in the limited follow-up period of six months; and a longer follow-up is necessary for ascertaining the relapse rates. The preliminary results however suggest that the addition of prothionamide to the standard WHO paucibacillary regimen is well-tolerated with increased inactivity rate and fewer instances of late reactions.