Untreated Leprosy Patients Research Articles

A comparative study on the detection of Mycobacterium leprae DNA in urine samples of leprosy patients using Rlep-PCR with other conventional samples.

Mycobacterium leprae causes leprosy that is highly stigmatized and chronic infectious skin disease. Only some diagnostic tools are being used for the identification M. leprae in clinical samples, such as bacillary detection, and histopathological tests. These methods are invasive and often have low sensitivity. Currently, the PCR technique has been used as an effective tool fordetecting M. leprae DNA across different clinical samples. The current study aims to detect M. leprae DNA in urine samples of untreated and treated leprosy patients using the Rlep gene (129 bp) and compared the detection among Ridley-Jopling Classification. Clinical samples (Blood, Urine, and Slit Skin Smears (SSS)) were collected from leprosy and Non-leprosy patients. DNA extraction was performed using standard laboratory protocol and Conventional PCR was carried out for all samples using Rlep gene target and the amplicons of urine samples were sequenced by Sanger sequencing to confirm the Rlep gene target. The M. leprae DNA was successfully detected in all clinical samples across all types of leprosy among all the study groups using RLEP-PCR. Rlep gene target was able to detect the presence of M. leprae DNA in 79.17% of urine, 58.33% of blood, and 50% of SSS samples of untreated Smear-Negative leprosy patients. The statistical significant difference (p = 0.004) was observed between BI Negative (Slit Skin Smear test) and RLEP PCR positivity in urine samples of untreated leprosy group. The PCR positivity using Rlep gene target (129 bp) was highest in all clinical samples among the study groups, across all types of leprosy. Untreated tuberculoid and PNL leprosy patients showed the highest PCR positivity in urine samples, indicating its potential as a non-invasive diagnostic tool for leprosy and even for contact screening.

Comparison of Serum Levels of Interleukin 17 and Interleukin 9 in Leprosy Patients with and without Lepra Reaction: A Cross-Sectional Analytical Study from Central India.

New pathways of host defence have emerged in leprosy, such as T helper (Th) -17, Th-9, T regulatory cells, and other factors like transforming growth factor-beta, etc. Interleukin (IL) 17 produced by Th17 cells has been found to be elevated in lepra reaction, especially type 2 lepra reaction (T2R). Role of IL-9 has not been studied widely in leprosy reactions so far. The study aimed to compare serum levels of IL-17 and IL-9 in leprosy patients with and without lepra reaction. This was a cross-sectional analytical study including untreated adult leprosy patients with and without lepra reaction. A total of 65 patients were included in the study with 30 leprosy patients without reaction and 35 with lepra reaction. Serum levels of IL-17 and IL-9 were measured in these patients using direct enzyme-linked immunosorbent assay and were compared. Borderline tuberculoid (BT) leprosy with type 1 and Lepromatous (LL) leprosy with T2R patients showed significantly higher levels of IL-17 than BT and LL leprosy patients without lepra reaction, respectively. LL patients with T2R showed significantly lower levels of IL-9 than lepromatous cases without reaction. IL-9 levels were higher in BT patients with T1R as compared to BT patients without reaction but the difference was not significant. We found evidence in support of role of IL-17 in the pathogenesis of T2R, which might serve as useful serum markers for the same. IL-17 might have a role in BT leprosy with T1R. IL-9 seems to have a protective role in T2R as opposed to IL-17, working in synergism with Th1 cytokines.

Downmodulation of Regulatory T Cells Producing TGF-β Participates in Pathogenesis of Leprosy Reactions.

Leprosy reactions are an acute and systemic manifestation, which occurs suddenly, can be severe and lead leprosy patients to disability. Reactional episodes are observed among half of the multibacillary patients, mainly in borderline lepromatous and lepromatous forms. They may begin at any time during multidrug therapy, and even before the treatment. Physical disabilities, which are the source of extreme suffering and pain for patients, occur in progression of the cellular immune response associated with a reaction and are still poorly understood. Thus, this work aimed to phenotypically and functionally characterize CD4+ and CD8+ Treg cells ex vivo and in response to Mycobacterium leprae (ML). We studied 52 individuals, including 18 newly diagnosed and untreated multibacillary leprosy patients, 19 reactional multibacillary patients (Type I or Type II episodes) and 15 healthy volunteers, included as controls, all residents of the city of Rio de Janeiro. The functional activity and frequencies of these cells were evaluated through multiparametric flow cytometry. In addition, the production of cytokines in supernatant from peripheral blood mononuclear cell cultures was also investigated against ML by enzyme-linked immunosorbent assay. Our results showed a decrease in CD4+TGF-β+ Treg and CD8+ TGF-β+ Treg in leprosy multibacillary patients during both types of reactional episodes. Alterations in the cytokine profile was also observed in Type II reactions, along with upregulation of IL-17 and IL-6 in supernatant. Thus, our study suggests that downregulation of Treg cells is related with both classes of reactional episodes, improving our understanding of immune hyporesponsiveness in multibacillary patients and hyperesponsiveness in both reactions.

Reduced vitamin D receptor (VDR) and cathelicidin antimicrobial peptide (CAMP) gene expression contribute to the maintenance of inflammatory immune response in leprosy patients

Leprosy is an infectious disease influenced by genetic, immunological, and environmental factors. Reduced gene expressions may be associated with the immunological response pattern and leprosy susceptibility. We investigated the direct and indirect effects of Vitamin D Receptor (VDR) and Cathelicidin Antimicrobial Peptide (CAMP) gene expressions on the serum levels of vitamin D, Cathelicidin, and cytokines in newly-diagnosed leprosy patients and post-six-months of multidrug therapy (MDT). Thirty-four leprosy patients were assessed, paucibacillary (PB; n = 14) and multibacillary (MB; n = 20) cases, untreated or having received six months of MDT, 18 healthy controls, and 25 household contacts. VDR and CAMP gene expression levels were strongly correlated to some important cytokines in both, untreated leprosy patients (PB, r = 0.9319; MB, r = 0.9569) and patients who had undergone MDT (PB, r = 0.9667; MB, r = 0.9569). We observed that both gene expressions directly influenced IL-2, IFN-γ, and IL-17F serum levels in leprosy patients compared to the household contacts and healthy individuals. VDR and CAMP gene expressions induced a persistent inflammatory response in PB and MB leprosy patients, even after six months of MDT, to fight the Mycobacterium leprae infection. Due to the persistent inflammatory profile, multidrug therapy is suggested to be maintained for more than six months, especially for MB patients. Vitamin D supplementation is recommended from the onset as a transcription factor to improve VDR and CAMP gene expression in leprosy patients.

BCG-induced immunity profiles in household contacts of leprosy patients differentiate between protection and disease

Leprosy is an infectious disease caused by Mycobacterium leprae leading to irreversible disabilities along with social exclusion. Leprosy is a spectral disease for which the clinical outcome after M. leprae infection is determined by host factors. The spectrum spans from anti-inflammatory T helper-2 (Th2) immunity concomitant with large numbers of bacteria as well as antibodies against M. leprae antigens in multibacillary (MB) leprosy, to paucibacillary (PB) leprosy characterised by strong pro-inflammatory, Th1 as well as Th17 immunity. Despite decades of availability of adequate antibiotic treatment, transmission of M. leprae is unabated. Since individuals with close and frequent contact with untreated leprosy patients are particularly at risk to develop the disease themselves, prophylactic strategies currently focus on household contacts of newly diagnosed patients.It has been shown that BCG (re)vaccination can reduce the risk of leprosy. However, BCG immunoprophylaxis in contacts of leprosy patients has also been reported to induce PB leprosy, indicating that BCG (re)vaccination may tip the balance between protective immunity and overactivation immunity causing skin/nerve tissue damage.In order to identify who is at risk of developing PB leprosy after BCG vaccination, amongst individuals who are chronically exposed to M. leprae, we analyzed innate and adaptive immune markers in whole blood of household contacts of newly diagnosed leprosy patients in Bangladesh, some of which received BCG vaccination. As controls, individuals from the same area without known contact with leprosy patients were similarly assessed.Our data show the added effect of BCG vaccination on immune markers on top of the effect already induced by M. leprae exposure. Moreover, we identified BCG-induced markers that differentiate between protective and disease prone immunity in those contacts.

Evaluation of Slit-Skin Smear Against Punch Biopsy in Diagnosing Leprosy: A Cross-sectional Study in a Tertiary Care Centre of West Bengal

Background: Leprosy is an infectious disease which faces diagnostic challenges. Slit-skin smear (SSS) is an age simple diagnostic technique, yet not commonly applied by health care providers. The study aimed to determine the effectiveness of SSS in terms of validity, diagnostic accuracy, and percentage agreement against punch biopsy in diagnosing leprosy among leprosy patients who were diagnosed with leprosy on clinical grounds only (i.e., number of skin lesions and/or peripheral nerve thickening). Methods: An evaluation study of diagnostic tests with a cross-sectional design was conducted at a tertiary care center of Bankura. In general, 70 new untreated leprosy patients, diagnosed solely by clinical grounds (i.e., count of skin lesions and/or thickening of the nerve) and attending the dermatology outpatient department (From February 2019 to January 2020) were enrolled in this study. After excluding pure neuritic, relapse, and seriously ill patients by consecutive sampling, they were subjected to both SSS and punch biopsy using a standard process. SPSS for Windows (Version 16.0., Chicago, SPSS Inc.) was used to analyze data. Z test, chi-square test, and kappa test were conducted to test the statistical significance between the effectiveness of SSS and punch biopsy. Results: The sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, negative likelihood ratio, diagnostic odds ratio (DOR), and diagnostic accuracy of SSS were 81.81%, 95.83%, 90%, 92%, 20%, 0.19%, 102.87%, and 91.42%, respectively. Based on the results, SSS could detect acid-fast bacilli (AFB) in clinically diagnosed leprosy cases slightly less than punch biopsy, but it was statistically insignificant (Z=0.3689, P=0.71138, df=1). Finally, Cohen’s Kappa coefficient was 0.796, representing substantial agreement between SSS and punch biopsy (95% CI: 0.641-0.951). Conclusions: Overall, SSS is more or less equally effective as compared to punch biopsy in confirming leprosy cases. Interest and training on SSS among resident doctors should be emphasized accordingly.

High rate of Mycobacterium leprae infection and nasal colonization among household contacts of previously undiagnosed leprosy patients

Objective This study examined the magnitude of M. leprae infection and nasal colonization among household contacts (HHCs) of newly diagnosed leprosy patients. Methods A community-based cross sectional study was conducted from July to October 2019 in Fedis district, eastern Ethiopia. This study involved all healthy HHCs of previously undiagnosed leprosy patients and equal numbers of randomly selected healthy endemic controls. All study participants underwent a standard physical examination. For leprosy-suspected HHCs a bacteriological examination was done. Nasal colonization among HHCs and endemic controls was evaluated for the presence of M. leprae-specific 500 bp repetitive elements (RLEP) using PCR. A structured questionnaire was used to obtain demographic, laboratory and leprosy-related clinical history. Results In this study, 48 HHCs of previously undiagnosed leprosy patients were evaluated for leprosy. Four new leprosy cases (co-prevalent) were confirmed, giving a detection rate of 8.3% (95%, CI = 2%, 19%); they were excluded from the HHCs study group. On PCR analysis, 5/44 (11.4%) (95% CI: 3%, 24%) HHCs and 3/15 (20%) (95% CI: 4%, 48%) untreated leprosy cases tested positive. Nasal secretion samples from all 44 healthy endemic controls tested negative by PCR. The positivity rate was significantly different among the study groups (P = 0.022) and disability grade is associated with PCR positivity (P = 0.003). However, nasal PCR positivity was not associated with the demographic characteristics of the study groups (p > 0.05). Conclusion This study provides evidence that HHCs of untreated leprosy patients have clinical infections and are involved in carriage of bacilli. Therefore, HHCs tracing is important to improve early diagnosis and decipher the transmission chain.

Presence of Senescent and Memory CD8+ Leukocytes as Immunocenescence Markers in Skin Lesions of Elderly Leprosy Patients.

Leprosy is an infectious disease that remains endemic in approximately 100 developing countries, where about 200,000 new cases are diagnosed each year. Moreover, multibacillary leprosy, the most contagious form of the disease, has been detected at continuously higher rates among Brazilian elderly people. Due to the so-called immunosenescence, characterized by several alterations in the quality of the immune response during aging, this group is more susceptible to infectious diseases. In view of such data, the purpose of our work was to investigate if age-related alterations in the immune response could influence the pathogenesis of leprosy. As such, we studied 87 individuals, 62 newly diagnosed and untreated leprosy patients distributed according to the age range and to the clinical forms of the disease and 25 healthy volunteers, who were studied as controls. The frequency of senescent and memory CD8+ leukocytes was assessed by immunofluorescence of biopsies from cutaneous lesions, while the serum levels of IgG anti-CMV antibodies were analyzed by chemiluminescence and the gene expression of T cell receptors' inhibitors by RT-qPCR. We noted an accumulation of memory CD8+ T lymphocytes, as well as reduced CD8+CD28+ cell expression in skin lesions from elderly patients, when compared to younger people. Alterations in LAG3 and PDCD1 gene expression in cutaneous lesions of young MB patients were also observed, when compared to elderly patients. Such data suggest that the age-related alterations of T lymphocyte subsets can facilitate the onset of leprosy in elderly patients, not to mention other chronic inflammatory diseases.

IL-6 promotes pathogenic Th17 in T1R leprosy reactions by stimulating Il17 producing by IL-6R pathway

Background: Leprosy is an infectious disease caused by Mycobacterium Leprae. Reversal reactions (RR) an inflammatory condition present as inflammation of local skin patches. Approximately, 30–40% of leprosy patients have undergone RR during the course of MDT. Previously, we have reported that IL-23 is involved in Th17 cell differentiation and not IL-6 in non-reactions leprosy patients. Subsequently, recent findings by our group on immunopathology of leprosy reactions showed that IL-6 induces Th17 differentiation together with TGF-β in leprosy reactions. As we asked the question that whether IL-6 or IL-23 induced Th17 cells are different in nature? Methods and materials: A total of 20 newly diagnosed and untreated stable leprosy and reactions patients were recruited. 48 h PBMCs cultures were established with a different combination of recombinants IL-6, IL-23, and TGF-β with or without Mycobacterium Leprae Sonicated Antigen. Subsequently, PBMCs cultures were blocked with either antagonized IL6R or IL23R antibodies. qPCR was used for gene expression analysis of IL-17A, IL17F, IL6R, and IL23R. Different phenotypes of Th17 cells were studied by flowcytometry and culture supernatant was estimated for cytokine ELISA. Results: In this study, leprosy reactions showed a high percentage of IL17A and IL17F producing CD4+ IL6R+ T cells as compared to stable leprosy patients (p < 0.001). On the other hand, leprosy reactions showed significant low (p < 0.001) IL-17A and F producing CD4+ IL23R+ Th17 cells as compared to stable leprosy patients in 48 h MLSA stimulated cultures. Furthermore, recombinant IL-6, IL23 and TGF-β significantly (p < 0.001) promotes IL17A and IL17F in CD4+ IL6+ T cells. Subsequently, IL6R and IL23R blocking experiments showed significantly (p < 0.01) down-regulated IL-17A and IL17F in T1R reaction as compared to stable leprosy. Conclusion: This study for the first time establishes that pathogenic Th17 cells IL-17 produce via IL-6R pathway in leprosy T1R reactions. Thus, present approaches that specifically target Th17 cells and/or the cytokines that promote their development, such as IL-6, TGF-β and IL-23A may provide more focused treatment strategies for the management of M. Leprae and its reaction. Hence, therapeutic approaches that aim to re-establish homeostasis by decreasing the production of IL-17 by Th17 may prove effective in the control of leprosy reaction.

Mycobacterium leprae deoxyribonucleic acid positivity on skin lesion of untreated leprosy patients and its route to the skin surface

The route of transmission of leprosy is still unclear. Multiple opinions exist regarding the environmental dissemination of Mycobacterium leprae (M. leprae). This research aims to identify the positive proportion of M. leprae deoxyribonucleic acid (DNA) on skin lesion surface of untreated multibacillary leprosy patients by polymerase chain reaction (PCR), and its association with the examination result of acid-fast bacilli (AFB) in epidermis, pilosebaceous unit, and sweat glands. The M. leprae DNA sample was taken from smears of the skin lesion surface by using PCR and the skin biopsy test was done to detect AFB in epidermis, pilosebaceous unit, and sweat glands. From 28 subjects, there was 82.1% positive proportion of M. leprae DNA on the skin lesion surface of untreated multibacillary leprosy patients, where all were found in the lepromatous leprosy and borderline leprosy type. The result of AFB was positive in epidermis (71.4%), pilosebaceous unit (46.4%) and sweat glands (53.6%). We found M. leprae by using PCR technique on skin lesion surface of untreated multibacillary leprosy patients, and its route which most likely was through the epidermis.

Mast cell heterogeneity and anti-inflammatory annexin A1 expression in leprosy skin lesions

Mast cells (MCs) have important immunoregulatory roles in skin inflammation. Annexin A1 (ANXA1) is an endogenous anti-inflammatory protein that can be expressed by mast cells, neutrophils, eosinophils, monocytes, epithelial and T cells. This study investigated MCs heterogeneity and ANXA1 expression in human dermatoses with special emphasis in leprosy. Sixty one skin biopsies from 2 groups were investigated: 40 newly diagnosed untreated leprosy patients (18 reaction-free, 11 type 1 reaction/T1R, 11 type 2 reaction/T2R); 21 patients with other dermatoses. Tryptase/try+ and chymase/chy + phenotypic markers and toluidine blue stained intact/degranulated MC counts/mm2 were evaluated. Try+/chy+ MCs and ANXA1 were identified by streptavidin-biotin-peroxidase immunostaining and density was reported. In leprosy, degranulated MCs outnumbered intact ones regardless of the leprosy form (from tuberculoid/TT to lepromatous/LL), leprosy reactions (reactional/reaction-free) and type of reaction (T1R/T2R). Compared to other dermatoses, leprosy skin lesions showed lower numbers of degranulated and intact MCs. Try+ MCs outnumbered chy+ in leprosy lesions (reaction-free/reactional, particularly in T2R), but not in other dermatoses. Compared to other dermatoses, ANXA1 expression, which is also expressed in mast cells, was higher in the epidermis of leprosy skin lesions, independently of reactional episode. In leprosy, higher MC degranulation and differential expression of try+/chy+ subsets independent of leprosy type and reaction suggest that the Mycobacterium leprae infection itself dictates the inflammatory MCs activation in skin lesions. Higher expression of ANXA1 in leprosy suggests its potential anti-inflammatory role to maintain homeostasis preventing tissue and nerve damage.

Enzymatic Oxidative Stress Indicators and Oxidative Stress Index in Patients of Leprosy

Introduction: Leprosy is a chronic granulomatous disease caused by Mycobacterium leprae. Oxidative stress caused by Reactive oxygen species (ROS) plays a crucial role in the pathogenesisis of leprosy.Objective: To measure the enzymatic oxidative stress indicators and oxidative stress index in patients of leprosy and compare them with healthy age &amp; sex matched controls.Materials and Methods: In this prospective study 30 untreated leprosy patients were included in the study and matched with 60 healthy controls. Biochemistry estimation was done with blood samples and MDA (lipid peroxidation), Nitric oxide (NO), (SOD) Superoxide dismutase (antioxidant enzyme), total oxidant status (TOS), total antioxidant status(TAS) and oxidative stress index (OSI) were estimated.Results: Highly significant rise (p&lt;0.0001) in serum MDA, NO, TOS and OSI was seen in leprosy patients when compared with controls with highly significant decline in SOD and TAS.Conclusion: The study confirms the oxidative stress in leprosy and suggests antioxidant therapy may be used as an adjuvant in the treatment of leprosy along with MDT.

Clinical and Histopathological Correlation in Hansen’s Disease

AIM OF THE STUDY: To assess, classify and adopt apt treatment protocols based on histopathological evalaution of leprosy (Hansen’s) patients. MATERIALS AND METHODS: The study was conducted on the skin biopsies of 35 patients newly registered at the Department of Dermatology, Venerology & Leprosy and subsequently reported at the histopathology section of the Department of Pathology, Coimbatore Medical College & Hospital, Coimbatore for a period of 18 months from May 2010 to October 2011. Skin biopsies of newly diagnosed, untreated leprosy patients were fixed in 10% formalin and routine histopathological examination done followed by special stain (Fite – Faraco) to evaluate the bacterial index (BI). OBSERVATION AND RESULTS: From the study it was observed that, the commonest age group affected by leprosy was 31 - 50 years, males are 2 times more commonly affected than females. (M : F = 1.9 :1) & the most commonest clinically diagnosed spectrum was tuberculoid leprosy (TT). The commonest histopathologically diagnosed spectrum was IL (34.28%), followed by TT & histoid leprosy. It was observed that there was complete agreement between clinical diagnosis and histopathological diagnosis in 62.85% cases and disagreement was observed in 37.15% cases. CONCLUSION: If there is discrepancy between clinical and histopathological classification, both the findings should be reviewed by independent experienced observers. In case of confirmed discrepancy, the more advanced findings (ie. towards the lepromatous pole) should be given greater weightage and the case is to be classified and treated accordingly. In all cases of leprosy, in order to type the exact spectrum of disease for appropriate treatment, skin biopsy of the lesions with histopathological examination followed by special stain (Fite -Faraco) is recommended.

Genomic diversity in Mycobacterium leprae isolates from leprosy cases in South India

ObjectiveThe Objective of this study was to identify the strain diversity of Mycobacterium leprae in terms of SNP types and subtypes stratified as per genomic single nucleotide polymorphisms, in clinical isolates of leprosy patients from a tertiary care leprosy center in South India. Further, the associations of SNP types with clinical outcomes in leprosy were also investigated. MethodsDNA was extracted from excisional skin biopsies of a total of 172 newly diagnosed untreated leprosy patients from a clinic in Tamil Nadu, in south India, that also serves patients from neighboring states. All the leprosy patients were those who voluntarily reported at the clinic during the study period of one year i.e., 2015. Clinical and histopathological details were collected at diagnosis and leprosy was confirmed through bacteriological smear examination and PCR for M. leprae specific RLEP region. SNP types and subtypes were determined by PCR amplification and Sanger sequencing of PCR products. ResultsM. leprae specific RLEP gene amplification was achieved in 160 out of 172 patients. Among 160 specimens 118(73.75%) were type 1 and 42 (26.25%) were type 2 and on subtyping it was noted that 88/160 (55.00%) were 1D, 25/160 (15.62%) 1C, 5/160 (3.12%) 1A, 33/160 (20.62%) 2G and 9/160 (5.62%) were 2H. ConclusionOur results indicated that subtype 1D is predominant in the south Indian population. We also noted 2G, 1C and 1A in the patient sample tested. Additionally we identified subtype 2H for the first time in India.

English

AIM OF THE STUDY: To assess, classify and adopt apt treatment protocols based on histopathological evalaution of leprosy (Hansen’s) patients. MATERIALS AND METHODS: The study was conducted on the skin biopsies of 35 patients newly registered at the Department of Dermatology, Venerology & Leprosy and subsequently reported at the histopathology section of the Department of Pathology, Coimbatore Medical College & Hospital, Coimbatore for a period of 18 months from May 2010 to October 2011. Skin biopsies of newly diagnosed, untreated leprosy patients were fixed in 10% formalin and routine histopathological examination done followed by special stain (Fite – Faraco) to evaluate the bacterial index (BI). OBSERVATION AND RESULTS: From the study it was observed that, the commonest age group affected by leprosy was 31 - 50 years, males are 2 times more commonly affected than females. (M : F = 1.9 :1) & the most commonest clinically diagnosed spectrum was tuberculoid leprosy (TT). The commonest histopathologically diagnosed spectrum was IL (34.28%), followed by TT & histoid leprosy. It was observed that there was complete agreement between clinical diagnosis and histopathological diagnosis in 62.85% cases and disagreement was observed in 37.15% cases. CONCLUSION: If there is discrepancy between clinical and histopathological classification, both the findings should be reviewed by independent experienced observers. In case of confirmed discrepancy, the more advanced findings (ie. towards the lepromatous pole) should be given greater weightage and the case is to be classified and treated accordingly. In all cases of leprosy, in order to type the exact spectrum of disease for appropriate treatment, skin biopsy of the lesions with histopathological examination followed by special stain (Fite -Faraco) is recommended.

Study of Oxidative Stress in Different Forms of Leprosy.

Background:Leprosy is a chronic infectious disease caused by Mycobacterium leprae. India records the highest number of new leprosy cases in the world. Oxidative stress may play a significant role in leprosy.Aim:The aim of the study was to evaluate oxidative stress in various forms of leprosy and compared to healthy controls.Materials and Methods:Seventy newly diagnosed, untreated leprosy patients were selected as cases and sixty healthy controls. Oxidative stress was evaluated by measuring serum malondialdehyde (MDA) level and superoxide dismutase (SOD) activity. Student's unpaired t-test and Anova (Analysis of Variance) test were used for analysis of data. P < 0.05 was considered as significant.Results:There was a statistically significant increase in the mean values of serum MDA level, MDA/SOD and a decrease in serum SOD activity in cases when compared to controls and the values were significantly associated with increased duration, bacterial load and multibacillary type in leprosy.Conclusion:Our study suggests that there was oxidative stress in leprosy. This warrants antioxidant supplementation to prevent tissue injury.

Serum Th1/Th2 and macrophage lineage cytokines in leprosy; correlation with circulating CD4(+) CD25(high) FoxP3(+) T-regs cells.

Not only macrophages, T-helper (Th)1 and Th2, but also CD4(+) CD25(high) FoxP3(+) regulatory T cells (T-regs) are involved in immune response to Mycobacterium leprae. We aimed to evaluate serum interleukin (IL)-1β and IL-12p70 (macrophage cytokines), interferon-γ (IFN-γ) (Th1 cytokine), IL-4 (Th2 cytokine) and circulating CD4(+) CD25(high) FoxP3(+) T-regs, in untreated leprosy patients. Forty three patients and 40 controls were assessed for the mentioned cytokines using ELISA. Patients were assessed for circulating T-regs using flow cytometry. Patients were subgrouped into tuberculoid (TT), pure neural leprosy (PNL), borderline cases, lepromatous (LL), type 1 reactional leprosy (RL1) and erythema nodosum leprosum (ENL). Serum IL-12p70, IFN-γ and IL-4 were significantly higher in patients versus controls (P<0.05). Serum IL-4 was highest in LL and lowest in RL1 (P=0.003). Serum IL-1β levels was significantly higher in multibacillary versus paucibacillary patients (P=0.006). Significantly higher T-regs levels was detected in TT, RL1 and PNL, while the lowest levels in ENL(P<0.001), with significant differences versus controls (P<0.05). FoxP3 expression% was significantly lower in PNL than other patients and controls (P<0.05). T-regs/T-effs was lowest in ENL(P<0.05). IFN-γ correlated positively with T-regs but negatively with IL-1β (P=0.041&0.046 respectively), which correlated positively with T-effs%( P=0.05). IL-4 correlated positively with T-regs FoxP3 expression% (P=0.009). We concluded that: Circulating T-regs were increased in TT, RL1 and PNL patients, known of relatively high cell-mediated immunity. This finding was supported by low FoxP3 expression (in PNL) and correlation between T-regs count and IFN-γ level. Overproduction of IL-4 in LL may infer liability to develop ENL, with disease progression and immune hyperactivation, marked by deficient T-regs and increased T-regs FoxP3 expression%. IL-1β probably has a pro-inflammatory role in multibacillary patients as correlated with T-effs%.

Serum Th17 cytokines in leprosy: correlation with circulating CD4(+) CD25 (high)FoxP3 (+) T-regs cells, as well as down regulatory cytokines.

Leprosy is not only a bacteriological disease but also an immunological disease, in which T helper17 and CD4(+) CD25(high)FoxP3(+) regulatory T cells (T-regs), among others, may play a role. We aimed to evaluate serum levels of interleukin (IL)-17, IL-22 (Th17 cytokines), IL-10 and transforming growth factor (TGF)-β (down regulatory cytokines) in 43 untreated leprosy patients and 40 controls by enzyme-linked immunosorbent assay, and to assess circulating CD4(+) CD25(high)FoxP3(+)T-regs in patients using flow cytometry. Patients were grouped into tuberculoid, pure neural, borderline, lepromatous, type 1 reactional leprosy, and erythema nodosum leprosum. IL-10 and TGF-β were significantly higher in patients as compared to controls (p<0.001), while IL-17, but not IL-22, was significantly lower (p<0.001), with no significant difference comparing patients' subgroups. Significantly higher CD4(+) CD25(high)FoxP3(+)T-regs levels was detected in tuberculoid, type 1 reaction and pure neural leprosy, while the lowest levels in erythema nodosum leprosum (p<0.001). TregsFoxP3 expression% was significantly lower in pure neural leprosy than other patients' subgroups (p<0.05). T-regs/T-effs was lowest in erythema nodosum leprosum (p<0.05). TGF-β correlated negatively with TregsFoxP3 expression% and T-effs% (p=0.009 and 0.018 respectively). Leprosy is associated with defective IL-17 and overproduction of IL-10 and TGF-β. Tuberculoid, type 1 reaction and pure neural leprosy express significantly higher circulating T-regs, consistent with effector immune mechanisms activation, but with lower TregsFoxP3 expression (in pure neural leprosy). Erythema nodosum leprosum is characterized by deficient T-regs and increased TregsFoxP3 expression%. The present study pinpointed a potential role of Th17, CD4(+) CD25(high)FoxP3(+)T-regs, and probably CD4(+) CD25(+)IL-10(+) T regulatory cells 1 (Tr1), and Th3 in leprosy.

Mycobacterium leprae DNA in peripheral blood may indicate a bacilli migration route and high-risk for leprosy onset

Leprosy epidemiological studies have been restricted to Mycobacterium leprae DNA detection in nasal and oral mucosa samples with scarce literature on peripheral blood. We present the largest study applying quantitative real-time PCR (qPCR) for the detection of M. leprae DNA in peripheral blood samples of 200 untreated leprosy patients and 826 household contacts, with results associated with clinical and laboratory parameters. To detect M. leprae DNA a TaqMan qPCR assay targeting the M. leprae ML0024 genomic region was performed. The ML0024 qPCR in blood samples detected the presence of bacillus DNA in 22.0% (44/200) of the leprosy patients: 23.2% (16/69) in paucibacillary (PB), and 21.4% (28/131) in multibacillary (MB) patients. Overall positivity among contacts was 1.2% (10/826), with similar percentages regardless of whether the index case was PB or MB. After a follow-up period of 7 years, 26 contacts have developed leprosy. Comparing the results of healthy contacts with those that become ill, ML0024 qPCR positivity at the time of diagnosis of their index case represented an impressive 14.78-fold greater risk for leprosy onset (95% CI 3.6–60.8; p <0.0001). In brief, contacts with positive PCR in blood at diagnosis of index cases are at higher risk of later leprosy onset and this marker might be combined with other prognostic markers for management of contacts, which requires further studies.

Synergistic antigen combinations for the development of interferon gamma release assays for paucibacillary leprosy

The development of immunodiagnostic tests for paucibacillary leprosy (PB) is based on Mycobacterium leprae specific-cell mediated immunity (CMI)/IFN-γ production. Recently, novel M. leprae protein antigens that stimulate CMI have been described. This study evaluated different M. leprae antigen combinations in whole blood assay (WBA). Five study groups were tested (20 per group): newly diagnosed, untreated PB patients and multibacillary leprosy patients (MB); household contacts of MB patients (HHC); healthy endemic controls (EC); pulmonary tuberculosis patients (TB). WBA (heparinized, 24 h 37 °C 5% CO₂) were stimulated with: 10 μg/ml of each individual M. leprae recombinant protein (rML) and five combinations of rML (46f + LID-1, ML0276 + LID-1, ML2055 + ML1632 + ML2044, ML0276 + 46f, ML2055 + LID-1)-M. leprae cell sonicate (MLCS, 10 μg/ml), PHA (1 μg/ml), and PBS alone. Human IFN-γ ELISA (QuantiFERON-TB Gold/QFT-G, Cellestis) was performed using stimulated plasma (arbitrary cut-off = 50 pg/ml). Three out of five antigen combinations (46f + LID-1, ML0276 + LID-1, ML2055 + ML1632 + ML2044) were able to increase the levels of IFN-γ production in WBA in a larger number of responders among both PB leprosy and contacts. However, the magnitude of IFN-γ responses was higher among contacts. The antigen combination (46f + ML0276) stimulated IFN-γ only in symptomatic PB leprosy patients and not in asymptomatic contacts. Few controls (EC, TB) responded to combinations (0-15%), indicating the specificity of the response in an endemic area with high BCG coverage. The synergistic effect of new combinations of M. leprae proteins upon IFN-γ production in WBA indicates their potential use for the development of an interferon gamma release assay/IGRA for the diagnosis of PB leprosy.