Specialized active leprosy search strategies in an endemic area of the Brazilian Amazon identifies a hypermutated Mycobacterium leprae strain causing primary drug resistance.

Introduction: Leprosy, an infectious disease caused by Mycobacterium leprae, remains a public health concern in endemic countries, particularly in Brazil. In this study, we conducted an active surveillance campaign in the hyperendemic city of Castanhal in the northeastern part of the state of Pará using clinical signs and symptoms combined with serological and molecular tools to diagnose new cases and to identify drug resistance of circulating M. leprae strains and their distribution in the community. Methods: During an active surveillance of one week, we enrolled 318 individuals using three different strategies to enroll subjects for this study: (i) an active survey of previously treated cases from 2006 to 2016 found in the Brazil National Notifiable Disease Information System database (n = 23) and their healthy household contacts (HHC) (n = 57); (ii) an active survey of school children (SC) from two primary public schools in low-income neighborhoods (n = 178), followed by visits to the houses of these newly diagnosed SC (n = 7) to examine their HHC (n = 34) where we diagnosed additional new cases (n = 6); (iii) and those people who spontaneously presented themselves to our team or the local health center with clinical signs and/or symptoms of leprosy (n = 6) with subsequent follow-up of their HHC when the case was confirmed (n = 20) where we diagnosed two additional cases (n = 2). Individuals received a dermato-neurological examination, 5 ml of peripheral blood was collected to assess the anti-PGL-I titer by ELISA and intradermal earlobe skin scrapings were taken from HHC and cases for amplification of the M. leprae RLEP region by qPCR. Results: Anti-PGL-I positivity was highest in the new leprosy case group (52%) followed by the treated group (40.9%), HHC (40%) and lowest in SC (24.6%). RLEP qPCR from SSS was performed on 124 individuals, 22 in treated cases, 24 in newly diagnosed leprosy cases, and 78 in HHC. We detected 29.0% (36/124) positivity overall in this sample set. The positivity in treated cases was 31.8% (7/22), while in newly diagnosed leprosy cases the number of positives were higher, 45.8% (11/23) and lower in HHC at 23.7% (18/76). Whole genome sequencing of M. leprae from biopsies of three infected individuals from one extended family revealed a hypermutated M. leprae strain in an unusual case of primary drug resistance while the other two strains were drug sensitive. Discussion: This study represents the extent of leprosy in an active surveillance campaign during a single week in the city of Castanhal, a city that we have previously surveyed several times during the past ten years. Our results indicate the continuing high transmission of leprosy that includes fairly high rates of new cases detected in children indicating recent spread by multiple foci of infection in the community. An unusual case of a hypermutated M. leprae strain in a case of primary drug resistance was discovered. It also revealed a high hidden prevalence of overt disease and subclinical infection that remains a challenge for correct clinical diagnosis by signs and symptoms that may be aided using adjunct laboratory tests, such as RLEP qPCR and anti-PGL-I serology.

Leprosy among children in an area without primary health care coverage in Caratateua Island, Brazilian Amazon.

Introduction: The detection of leprosy in children is an important epidemiological marker of the disease, indicating the community's early exposure to Mycobacterium leprae and active transmission of the infection. Methods: In order to detect new cases among children by combining clinical evaluation and laboratory tests, we conducted an active case finding among individuals under 15 years old on Caratateua Island, located in the city of Belém, in the Pará state, an endemic region in the Amazon. Dermato-neurological examination, collection of 5 mL of peripheral blood for IgM anti-PGL-I antibody titration, and intradermal scraping for bacilloscopy and amplification of the specific RLEP region by qPCR were performed. Results: Out of the 56 examined children, 28/56 (50%) new cases were identified. At the time of evaluation, 38/56 (67.8%) children presented one or more clinical alterations. Seropositivity was detected in 7/27 (25.9%) new cases and 5/24 (20.8%) undiagnosed children. DNA amplification of Mycobacterium leprae was observed in 23/28 (82.1%) of new cases and in 5/26 (19.2%) of non-cases. Out of the total cases, 11/28 (39.2%) were exclusively diagnosed by clinical evaluation performed during the active case finding. Seventeen new cases (60.8%) were detected considering the clinical alterations found in addition to positive results for qPCR. In this group, 3/17 (17.6%) qPCR-positive children presented significant clinical changes 5.5 months after the first evaluation. Discussion: Our research detected a number of cases 5.6 times higher compared to the total number of pediatric cases recorded throughout the year 2021 in the municipality of Belém, which shows a critical scenario of underdiagnosing of leprosy among children under 15 years old in the region. We propose the use of qPCR technique to identify new cases among children with oligosymptomatic or early disease in endemic areas, in addition to the training of Primary Health Care professionals and the implementation of the Family Health Strategy coverage in the visited area.

ELISA Test Based on the Phenolic Glycolipid-I (PGL-I) of Mycobacterium leprae: A Reality of a Laboratory from a Non-Endemic Country.

BACKGROUND: Leprosy is a neglected tropical disease caused by Mycobacterium leprae, leading to disabilities if untreated. The ELISA based on phenolic glycolipid I (PGL-I), or its synthetic version ND-O-BSA, is almost universally positive in multibacillary leprosy and thus extensively used in endemic countries. Household contacts with a positive antibody titer have ~6-fold higher probability to develop the disease than those with a negative titer. Thus, the aim of the study was to evaluate the performance of this ELISA in the setting of a non-endemic country. METHODS: We calculate the cut-off using optimized O.D. thresholds, generated by receiver operating characteristics (ROC) curve analysis, testing 39 well-characterized sera obtained from lepromatous leprosy patients with strongly positive ND-O-BSAELISA titer and 39 sera from healthy non-endemic patients never exposed to M. leprae or M. tuberculosis. Indeed, we tested a second set of sera from suspected or confirmed leprosy or household contacts (SLALT group, n=50), and patients with tuberculosis (control group, n=40). RESULTS: We detected 56.4% of SLALT and 22.5% of tuberculosis as positive, consistent with the literature. CONCLUSION: The ELISA based on ND-O-BSA may thus be considered a good option to be used in a non-endemic area as a screening tool in at risk population usually coming to our center.

Semmes-Weinstein monofilament: A tool to quantify skin sensation in macular lesions for leprosy diagnosis.

INTRODUCTION: Hypochromatic macules with altered sensitivity are the first manifestations of skin leprosy. Validation of this sensory loss assists in the confirmation of the clinical diagnosis. AIMS: The aim of the study was to quantify the loss of sensation in leprosy lesions using the Semmes-Weinstein monofilament to strengthen the clinical diagnosis mainly of macular forms. METHODS: Seventy-four hypochromatic macules in the macular leprosy subgroup, 27 typical borderline leprosy subgroup lesions and 49 macules of other macular dermatoses (non-leprosy group) were evaluated using the 0.05 g force Semmes-Weinstein monofilament to quantify the alteration of sensitivity within and outside of the lesions. The esthesiometric change index was established as the total number of points with altered sensation divided by the total number of tested points within the lesions to calculate the internal esthesiometric change index and outside the lesions to calculate the peripheral esthesiometric change index; these indexes were calculated for all groups. The difference (Δ) between the esthesiometric change indices of the lesional area and the adjacent skin was calculated for the leprosy and nonleprosy groups. RESULTS: The percentage of points with touch sensitivity alterations within the macular and typical borderline leprosy lesions was higher in leprosy than in the non-leprosy group. The borderline and macular leprosy presented higher esthesiometric change index within injured areas than outside injured areas or in the nonleprosy group (P < 0.005). When internal esthesiometric change index values in the macular and borderline leprosy groups were higher than 0.53 and 0.5, respectively, the receiver operating characteristic curve showed 98% sensitivity and approximately 99% specificity for both groups (P < 0.0001). Regarding the difference between indices, borderline and macular leprosy had values that were higher and closer to one than in the nonleprosy group (P < 0.0001), with 100% sensitivity and 96.5% specificity for leprosy diagnosis when ΔLG was higher than 0.34. A limitation was the inability to perform a double-blind study. CONCLUSION: Semmes-Weinstein esthesiometry is a simple, useful and low-cost tool to quantify the focal alteration of cutaneous sensitivity to improve clinical leprosy diagnosis, especially for macular lesions.

Latent leprosy infection identified by dual RLEP and anti-PGL-I positivity: Implications for new control strategies.

The number of new cases of leprosy reported worldwide has remained essentially unchanged for the last decade despite continued global use of free multidrug therapy (MDT) provided to any diagnosed leprosy patient. In order to more effectively interrupt the chain of transmission, new strategies will be required to detect those with latent disease who contribute to furthering transmission. To improve the ability to diagnose leprosy earlier in asymptomatic infected individuals, we examined the combined use of two well-known biomarkers of M. leprae infection, namely the presence of M. leprae DNA by PCR from earlobe slit skin smears (SSS) and positive antibody titers to the M. leprae-specific antigen, Phenolic Glycolipid I (anti-PGL-I) from leprosy patients and household contacts living in seven hyperendemic cities in the northern state of Pará, Brazilian Amazon. Combining both tests increased sensitivity, specificity and accuracy over either test alone. A total of 466 individuals were evaluated, including 87 newly diagnosed leprosy patients, 52 post-treated patients, 296 household contacts and 31 healthy endemic controls. The highest frequency of double positives (PGL-I+/RLEP+) were detected in the new case group (40/87, 46%) with lower numbers for treated (12/52, 23.1%), household contacts (46/296, 15.5%) and healthy endemic controls (0/31, 0%). The frequencies in these groups were reversed for double negatives (PGL-I-/RLEP-) for new cases (6/87, 6.9%), treated leprosy cases (15/52, 28.8%) and the highest in household contacts (108/296, 36.5%) and healthy endemic controls (24/31, 77.4%). The data strongly suggest that household contacts that are double positive have latent disease, are likely contributing to shedding and transmission of disease to their close contacts and are at the highest risk of progressing to clinical disease. Proposed strategies to reduce leprosy transmission in highly endemic areas may include chemoprophylactic treatment of this group of individuals to stop the spread of bacilli to eventually lower new case detection rates in these areas.

Leprosy in a prison population: A new active search strategy and a prospective clinical analysis.

BACKGROUND: This study evaluates an active search strategy for leprosy diagnosis based on responses to a Leprosy Suspicion Questionnaire (LSQ), and analyzing the clinical, immunoepidemiological and follow-up aspects for individuals living in a prison population. METHODS: A cross-sectional study based on a questionnaire posing 14 questions about leprosy symptoms and signs that was distributed to 1,400 prisoners. This was followed by dermatoneurological examination, anti-PGL-I serology and RLEP-PCR. Those without leprosy were placed in the Non-leprosy Group (NLG, n = 1,216) and those diagnosed with clinical symptoms of leprosy were placed in the Leprosy Group (LG, n = 34). FINDINGS: In total, 896 LSQ were returned (64%), and 187 (20.9%) of the responses were deemed as positive for signs/symptoms, answering 2.7 questions on average. Clinically, 1,250 (89.3%) of the prisoners were evaluated resulting in the diagnosis of 34 new cases (LG), based on well-accepted clinical signs and symptoms, a new case detection rate of 2.7% within this population, while the NLG were comprised of 1,216 individuals. The confinement time medians were 39 months in the LG while it was 36 months in the NLG (p>0.05). The 31 leprosy cases who responded to the questionnaire (LSQ+) had an average of 1.5 responses. The symptoms "anesthetized skin area" and "pain in nerves" were most commonly mentioned in the LG while "tingling, numbness in the hands/feet", "sensation of pricks and needles", "pain in nerves" and "spots on the skin" responses were found in more than 30% of questionnaires in the NLG. Clinically, 88.2% had dysesthetic macular skin lesions and 97.1% presented some peripheral nerve impairment, 71.9% with some degree of disability. All cases were multibacillary, confirming a late diagnosis. Anti-PGL-I results in the LG were higher than in the NLG (p<0.0001), while the RLEP-PCR was positive in 11.8% of the patients. INTERPRETATION: Our findings within the penitentiary demonstrated a hidden prevalence of leprosy, although the individuals diagnosed were likely infected while living in their former communities and not as a result of exposure in the prison. The LSQ proved to be an important screening tool to help identify leprosy cases in prisons.

Leprosy piRnome: exploring new possibilities for an old disease.

Leprosy, which is caused by the human pathogen Mycobacterium leprae, causes nerve damage, deformity and disability in over 200,000 people every year. Because of the long doubling time of M. leprae (13 days) and the delayed onset of detectable symptoms, which is estimated to be approximately 3-7 years after infection, there is always a large percentage of subclinically infected individuals in the population who will eventually develop the disease, mainly in endemic countries. piRNAs comprise the largest group of small noncoding RNAs found in humans, and they are distinct from microRNAs (miRNAs) and small interfering RNAs (siRNAs). piRNAs function in transposon silencing, epigenetic regulation, and germline development. The functional role of piRNAs and their associated PIWI proteins have started to emerge in the development of human cancers and viral infections, but their relevance to bacterial diseases has not been investigated. The present study reports the piRNome of human skin, revealing that all but one of the piRNAs examined are downregulated in leprosy skin lesions. Considering that one of the best characterized functions of piRNAs in humans is posttranscriptional mRNA silencing, their functions are similar to what we have described for miRNAs, including acting on apoptosis, M. leprae recognition and engulfment, Schwann cell (SC) demyelination, epithelial-mesenchymal transition (EMT), loss of sensation and neuropathic pain. In addition to new findings on leprosy physiopathology, the discovery of relevant piRNAs involved in disease processes in human skin may provide new clues for therapeutic targets, specifically to control nerve damage, a prominent feature of leprosy that has no currently available pharmaceutical treatment.

The immunology of other mycobacteria: M. ulcerans, M. leprae.

Mycobacterial pathogens can be categorized into three broad groups: Mycobacterium tuberculosis complex causing tuberculosis, M. leprae and M. lepromatosis causing leprosy, and atypical mycobacteria, or non-tuberculous mycobacteria (NTM), responsible for a wide range of diseases. Among the NTMs, M. ulcerans is responsible for the neglected tropical skin disease Buruli ulcer (BU). Most pathogenic mycobacteria, including M. leprae, evade effector mechanisms of the humoral immune system by hiding and replicating inside host cells and are furthermore excellent modulators of host immune responses. In contrast, M. ulcerans replicates predominantly extracellularly, sheltered from host immune responses through the cytotoxic and immunosuppressive effects of mycolactone, a macrolide produced by the bacteria. In the year 2018, 208,613 new cases of leprosy and 2713 new cases of BU were reported to WHO, figures which are notoriously skewed by vast underreporting of these diseases.

miRNome Expression Analysis Reveals New Players on Leprosy Immune Physiopathology.

Leprosy remains as a public health problem and its physiopathology is still not fully understood. MicroRNAs (miRNA) are small RNA non-coding that can interfere with mRNA to regulate gene expression. A few studies using DNA chip microarrays have explored the expression of miRNA in leprosy patients using a predetermined set of genes as targets, providing interesting findings regarding the regulation of immune genes. However, using a predetermined set of genes restricted the possibility of finding new miRNAs that might be involved in different mechanisms of disease. Thus, we examined the miRNome of tuberculoid (TT) and lepromatous (LL) patients using both blood and lesional biopsies from classical leprosy patients (LP) who visited the Dr. Marcello Candia Reference Unit in Sanitary Dermatology in the State of Pará and compared them with healthy subjects. Using a set of tools to correlate significantly differentially expressed miRNAs with their gene targets, we identified possible interactions and networks of miRNAs that might be involved in leprosy immunophysiopathology. Using this approach, we showed that the leprosy miRNA profile in blood is distinct from that in lesional skin as well as that four main groups of genes are the targets of leprosy miRNA: (1) recognition and phagocytosis, with activation of immune effector cells, where the immunosuppressant profile of LL and immunoresponsive profile of TT are clearly affected by miRNA expression; (2) apoptosis, with supportive data for an antiapoptotic leprosy profile based on BCL2, MCL1, and CASP8 expression; (3) Schwann cells (SCs), demyelination and epithelial-mesenchymal transition (EMT), supporting a role for different developmental or differentiation gene families, such as Sox, Zeb, and Hox; and (4) loss of sensation and neuropathic pain, revealing that RHOA, ROCK1, SIGMAR1, and aquaporin-1 (AQP1) may be involved in the loss of sensation or leprosy pain, indicating possible new therapeutic targets. Additionally, AQP1 may also be involved in skin dryness and loss of elasticity, which are well known signs of leprosy but with unrecognized physiopathology. In sum, miRNA expression reveals new aspects of leprosy immunophysiopathology, especially on the regulation of the immune system, apoptosis, SC demyelination, EMT, and neuropathic pain.

Evidence of hidden leprosy in a supposedly low endemic area of Brazil.

OBJECTIVES: Show that hidden endemic leprosy exists in a municipality of inner São Paulo state (Brazil) with active surveillance actions based on clinical and immunological evaluations. METHODS: The study sample was composed by people randomly selected by a dermatologist during medical care in the public emergency department and by active surveillance carried out during two days at a mobile clinic. All subjects received a dermato-neurological examination and blood sampling to determine anti-PGL-I antibody titers by enzyme-linked immunosorbent assay (ELISA). RESULTS: From July to December 2015, 24 new cases of leprosy were diagnosed; all were classified as multibacillary (MB) leprosy, one with severe Lucio's phenomenon. Seventeen (75%) were found with grade-1 or 2 disability at the moment of diagnosis. Anti-PGL-I titer was positive in 31/133 (23.3%) individuals, only 6/24 (25%) were positive in newly diagnosed leprosy cases. CONCLUSIONS: During the last ten years before this study, the average new case detection rate (NCDR) in this town was 2.62/100,000 population. After our work, the NCDR was raised to 42.8/100,000. These results indicate a very high number of hidden leprosy cases in this supposedly low endemic area of Brazil.

Evidence of hidden leprosy in a supposedly low endemic area of Brazil

OBJECTIVES Show that hidden endemic leprosy exists in a municipality of inner São Paulo state (Brazil) with active surveillance actions based on clinical and immunological evaluations. METHODS The study sample was composed by people randomly selected by a dermatologist during medical care in the public emergency department and by active surveillance carried out during two days at a mobile clinic. All subjects received a dermato-neurological examination and blood sampling to determine anti-PGL-I antibody titers by enzyme-linked immunosorbent assay (ELISA). RESULTS From July to December 2015, 24 new cases of leprosy were diagnosed; all were classified as multibacillary (MB) leprosy, one with severe Lucio's phenomenon. Seventeen (75%) were found with grade-1 or 2 disability at the moment of diagnosis. Anti-PGL-I titer was positive in 31/133 (23.3%) individuals, only 6/24 (25%) were positive in newly diagnosed leprosy cases. CONCLUSIONS During the last ten years before this study, the average new case detection rate (NCDR) in this town was 2.62/100,000 population. After our work, the NCDR was raised to 42.8/100,000. These results indicate a very high number of hidden leprosy cases in this supposedly low endemic area of Brazil.

Leprosy in Children.

PURPOSE OF REVIEW: This manuscript aims to review the cutting-edge developments regarding to the diagnosis, management, and prevention of leprosy in children. RECENT FINDINGS: Leprosy transmission still occurs continuously in some endemic areas in the world. Leprosy in children below 15 years old is a robust indicator of active source of infection in the community where they live. A special focus on children to reduce disabilities and reduce transmission is one of the core areas of interventions of the global leprosy strategy 2016-2020. Ongoing research is trying to develop better diagnostic tests and to advance chemoprophylaxis and immunoprophylaxis approaches. Early diagnosis in children can be hard because of the wide range of clinical aspects of the skin lesions and mainly due to the difficulty of performing the clinical peripheral nerve evaluation. We must maintain leprosy expertise and improve the health professionals training for leprosy diagnosis, since we still have a long journey to reach leprosy elimination.

Spatial epidemiology and serologic cohorts increase the early detection of leprosy.

BACKGROUND: Leprosy remains an important public health problem in some specific high-burden pockets areas, including the Brazilian Amazon region, where it is hyperendemic among children. METHODS: We selected two elementary public schools located in areas most at risk (cluster of leprosy or hyperendemic census tract) to clinically evaluate their students. We also followed anti-PGL-I seropositive and seronegative individuals and households for 2 years to compare the incidence of leprosy in both groups. RESULTS: Leprosy was detected in 11 (8.2 %) of 134 school children in high risk areas. The difference in the prevalence was statistically significant (p < .05) compared to our previous findings in randomly selected schools (63/1592; 3.9 %). The 2-year follow-up results showed that 22.3 and 9.4 % of seropositive and seronegative individuals, respectively, developed leprosy (p = .027). The odds of developing overt disease in seropositive people were 2.7 times that of negative people (p < .01), indicating that a follow-up of 10 seropositives has a >90 % probability to detect at least one new case in 2 years. The odds of clinical leprosy were also higher in "positive houses" compared to "negative houses" (p < .05), indicating that a follow-up of ten people living in households with at least one seropositive dweller have a 85 % probability to detect at least one new case in 2 years. CONCLUSIONS: Targeted screening involving school-based surveillance planned using results obtained by spatial analysis and targeted household and individual continuous surveillance based on serologic data should be applied to increase the early detection of new leprosy cases.

Spatial analysis spotlighting early childhood leprosy transmission in a hyperendemic municipality of the Brazilian Amazon region.

BACKGROUND: More than 200,000 new cases of leprosy were reported by 105 countries in 2011. The disease is a public health problem in Brazil, particularly within high-burden pockets in the Amazon region where leprosy is hyperendemic among children. METHODOLOGY: We applied geographic information systems and spatial analysis to determine the spatio-temporal pattern of leprosy cases in a hyperendemic municipality of the Brazilian Amazon region (Castanhal). Moreover, we performed active surveillance to collect clinical, epidemiological and serological data of the household contacts of people affected by leprosy and school children in the general population. The occurrence of subclinical infection and overt disease among the evaluated individuals was correlated with the spatio-temporal pattern of leprosy. PRINCIPAL FINDINGS: The pattern of leprosy cases showed significant spatio-temporal heterogeneity (p<0.01). Considering 499 mapped cases, we found spatial clusters of high and low detection rates and spatial autocorrelation of individual cases at fine spatio-temporal scales. The relative risk of contracting leprosy in one specific cluster with a high detection rate is almost four times the risk in the areas of low detection rate (RR = 3.86; 95% CI = 2.26-6.59; p<0.0001). Eight new cases were detected among 302 evaluated household contacts: two living in areas of clusters of high detection rate and six in hyperendemic census tracts. Of 188 examined students, 134 (71.3%) lived in hyperendemic areas, 120 (63.8%) were dwelling less than 100 meters of at least one reported leprosy case, 125 (66.5%) showed immunological evidence (positive anti-PGL-I IgM titer) of subclinical infection, and 9 (4.8%) were diagnosed with leprosy (8 within 200 meters of a case living in the same area). CONCLUSIONS/SIGNIFICANCE: Spatial analysis provided a better understanding of the high rate of early childhood leprosy transmission in this region. These findings can be applied to guide leprosy control programs to target intervention to high risk areas.