Clinical and Imaging Characteristics of Smear Negative Pulmonary Tuberculosis Patients: A Comparative Study.

Background: Prevalence surveys in Ethiopia indicate smear negative pulmonary tuberculosis (SNPTB) taking the major share of the overall TB burden. It has also been a diagnostic dilemma worldwide leading to diagnostic delays and difficulty in monitoring treatment outcomes. This study determines and compares the clinical and imaging findings in SNPTB and smear positive PTB (SPPTB). Methodology. A case-control study was conducted on 313 PTB (173 SNPTB) patients. Data and sputum samples were collected from consented patients. Smear microscopy, GeneXpert, and culture analyses were performed on sputum samples. Data were analyzed using Stata version 17; a P value < 0.05 was considered statistically significant. Results: Of the 173 SNPTB patients, 42% were culture positive with discordances between test results reported by health facilities and Armauer Hansen Research Institute laboratory using concentrated smear microscopy. A previous history of TB and fewer cavitary lesions were significantly associated with SNPTB. Conclusions: Though overall clinical presentations of SNPTB patients resemble those seen in SPPTB patients, a prior history of TB was strongly associated with SNPTB. Subject to further investigations, the relatively higher discrepancies seen in TB diagnoses reflect the posed diagnostic challenges in SNPTB patients, as a higher proportion of these patients are also seen in Ethiopia.

Training and Active Case Detection to Prevent Leprosy: Effect on Knowledge, Attitude and Skills of Health Workers on Early Diagnosis of Leprosy in a Leprosy Hotspot District in Ethiopia.

BACKGROUND: Despite all of the efforts, leprosy continues to affect hundreds of thousands of people every year, including children, showing the ongoing transmission of the disease within the population. The transmission of leprosy can be interrupted through an integrated approach that includes active case-finding, contact tracing and capacity building of health workers. METHODS: A cross-sectional study design was used to assess the knowledge, attitudes and skills of health workers in the screening and diagnosis of leprosy. One hundred and eighty-one and eighty-eight health care workers participated in the pre-and post-assessment surveys, respectively. Data were collected through interviews and an observational checklist. Frequency tables and graphs were used to describe the study variables, and statistical significance between pre- and post-assessment surveys was declared at p-value < 0.5. RESULT: The percentages of healthcare workers with good knowledge, positive attitudes and skills were 61.2%, 55.6% and 51.7% in the pre-assessment survey and 77.3%, 56.3% and 75.0%, respectively, in the post-assessment survey. There was a significant improvement in the knowledge and skill scores of participants in the post-assessment survey (p < 0.01). During the campaign, 3780 index contacts were screened; 570 (15.1%) were diagnosed with skin diseases, and 17 new leprosy cases were diagnosed (case detection rate of 45 per 10,000 contacts). CONCLUSION: Training improved the knowledge and skills of healthcare workers, and a large number of skin diseases were detected through mass screening and active case findings. Providing training for frontline healthcare workers contributed to the detection of more cases and facilitated early detection of leprosy cases.

Public perceptions of genomic studies and hereditary diseases in Aari community, South Omo Zone, Ethiopia.

BACKGROUND: Genetic and genomic research is revolutionizing precision medicine; however, addressing ethical and cultural aspects is crucial to ensure ethical conduct and respect for community values and beliefs. This study explored the beliefs, perceptions and concerns of the Aari community in South Ethiopia regarding genetic concepts, hereditary diseases and ethical research practices related to sample collection, storage and sharing. METHODS: In-depth interviews and focus group discussions were conducted with community elders, health officials, tuberculosis patients and apparently healthy individuals. Data were thematically analysed using MAXQDA software. RESULTS: Participants identified diseases such as podoconiosis, leprosy, goitre and epilepsy as hereditary and perceived some as 'curses' due to generational impact and social stigma. Disease susceptibility was attributed to divine intervention or factors such as malnutrition and sanitation. Although hereditary diseases were considered unavoidable, in some cases environmental factors were acknowledged. Participants shared personal examples to demonstrate inheritance concepts. Blood held cultural significance, and concerns about its potential misuse resulted in scepticism towards giving samples. CONCLUSIONS: This study emphasizes the significance of comprehending local beliefs and perceptions and stresses the need to establish effective communication, build trust and address underlying causes of hesitancy to improve recruitment and ensure ethical conduct.

Reaching those at risk: Active case detection of leprosy and contact tracing at Kokosa, a hot spot district in Ethiopia.

INTRODUCTION: Leprosy is a chronic mycobacterial disease of public health importance. It is one of the leading causes of permanent physical disability. The prevalence of leprosy in Ethiopia has remained stagnant over the last decades. The aim of the study was to identify new leprosy cases and trace household contacts at risk of developing leprosy by active case detection. The study area was Kokosa district, West Arsi zone, Oromia region, Ethiopia. METHOD: A prospective longitudinal study was conducted from June 2016-September 2018 at Kokosa district. Ethical approvals were obtained from all relevant institutions. Health extension workers screened households by house-to-house visits. Blood samples were collected and the level of anti-PGL-I IgM measured at two-time points. RESULTS: More than 183,000 people living in Kokosa district were screened. Dermatologists and clinical nurses with special training on leprosy confirmed the new cases, and their household contacts were included in the study. Of the 91 new cases diagnosed and started treatment, 71 were recruited into our study. Sixty-two percent were males and 80.3% were multibacillary cases. A family history of leprosy was found in 29.6% of the patients with cohabitation ranging from 10 to 30 years. Eight new leprosy cases were diagnosed among the 308 household contacts and put on multi-drug therapy. The New Case Detection Rate increased from 28.3/100,000 to 48.3/100,000 between 2015/2016 and 2016/2017. Seventy one percent of leprosy patients and 81% of the household contacts' level of anti-PGL-I IgM decreased after treatment. In conclusion,the results of the study showed the importance of active case detection and household contact tracing. It enhances early case finding, and promotes early treatment, thereby interrupting transmission and preventing potential disability from leprosy.

Changes in plasma levels of endocrine hormones in lepromatous leprosy patients.

Background: Leprosy affects various endocrine glands and causes disorders in internal organs in addition to the skin and peripheral nerves. These disorders are often silent and remain undiagnosed or underreported. In particular, patterns of hormone changes during leprosy, especially in lepromatous leprosy (LL) patients, are often associated with dysregulation of different endocrine and sex hormones. The aim of this study was to assess changes in four endocrine hormones - namely cortisol, dehydroepiandrosterone (DHEA), growth hormone (GH), and leptin - among LL patients compared with apparently healthy controls. Method: In total, 80 plasma samples were systematically retrieved from a biorepository at the Armauer Hansen Research Institute (AHRI), based on quality, adequacy of sample volume, and appropriateness of linked clinical and sociodemographic data. Forty of the samples were obtained from LL patients (cases) and the remaining 40 from apparently healthy controls. Enzyme-linked immunosorbant assay (ELISA) was used to quantify levels of DHEA, cortisol, GH, and leptin hormones in the plasma samples. Data were analyzed using non-parametric statistics and the Mann-Whitney U-test (GraphPad Prism version 7.01). A p-value < 0.05 was considered statistically significant. Results: Plasma levels of cortisol concentration were significantly higher in LL cases (median = 111.4 ng/ml, range = 20.54-525.7) compared with healthy controls (median = 51.98 ng/ml, range = 3.805-328.4) (p = 0.003). Levels of GH and leptin were significantly lower in LL cases compared with healthy controls (median values for GH = 1.01 µIU/ml, range = 0.4625-86.82 and 2 µIU/ml, range = 0.5838-63.36, respectively (p = 0.022); median values for leptin = 891 pg/ml, range = 728.4-21816 and 5147 pg/ml, range = 730.4-52747, respectively (p < 0.0001)). There was an apparent reduction in the plasma levels of DHEA among LL cases compared with healthy controls (p = 0.297), although this difference was not statistically significant. Conclusion: Alterations in levels of endocrine hormones seen in LL patients reflect clinical and immunological conditions during lepromatous leprosy. However, large-scale studies are warranted to determine how leprosy causes such alterations in hormones and the interplay between endocrine hormones and the immune system during leprosy disease.

Prolonged delays in leprosy case detection in a leprosy hot spot setting in Eastern Ethiopia.

BACKGROUND: Leprosy or Hansen's disease is known to cause disability and disfigurement. A delay in case detection of leprosy patients can lead to severe outcomes. In Ethiopia, the disability rates caused by leprosy among new cases are relatively high compared to other endemic countries. This suggests the existence of hidden leprosy cases in the community and a delay in timely detection. To reduce disability rates, it is crucial to identify the factors associated with this delay. This study aimed to determine the extent of delay in case detection among leprosy cases in Eastern Ethiopia. METHODS: This cross-sectional explorative study was conducted in January and February 2019 among 100 leprosy patients diagnosed ≤6 months prior to inclusion. A structured questionnaire was used to collect data, including the initial onset of symptoms, and the reasons for delayed diagnosis. Descriptive statistics, including percentages and medians, were used to describe the case detection delay. Logistic regression analysis was carried out to evaluate the predictors of delay in case detection of >12 months. FINDINGS: The median age of patients was 35 years, with a range of 7 to 72 years. The majority were male (80%) and rural residents (90%). The median delay in case detection was 12 months (interquartile range 10-36 months) among the included patients. The mean delay in case detection was 22 months, with a maximum delay of 96 months. The overall prevalence of disability among the study population was 42% (12% grade I and 30% grade II). Fear of stigma (p = 0.018) and experiencing painless symptoms (p = 0.018) were highly associated with a delay in case detection of >12 months. CONCLUSIONS: Being afraid of stigma and having painless symptoms, which are often misinterpreted as non-alarming at the onset of the disease, were associated with a delay in case detection. This study showed the need to increase knowledge on early symptoms of leprosy among affected communities. Furthermore, it is important to support initiatives that reduce leprosy related stigma and promote health worker training in leprosy control activities.

Regulatory T cells in erythema nodosum leprosum maintain anti-inflammatory function.

BACKGROUND: The numbers of circulating regulatory T cells (Tregs) are increased in lepromatous leprosy (LL) but reduced in erythema nodosum leprosum (ENL), the inflammatory complication of LL. It is unclear whether the suppressive function of Tregs is intact in both these conditions. METHODS: A longitudinal study recruited participants at ALERT Hospital, Ethiopia. Peripheral blood samples were obtained before and after 24 weeks of prednisolone treatment for ENL and multidrug therapy (MDT) for participants with LL. We evaluated the suppressive function of Tregs in the peripheral blood mononuclear cells (PBMCs) of participants with LL and ENL by analysis of TNFα, IFNγ and IL-10 responses to Mycobacterium leprae (M. leprae) stimulation before and after depletion of CD25+ cells. RESULTS: 30 LL participants with ENL and 30 LL participants without ENL were recruited. The depletion of CD25+ cells from PBMCs was associated with enhanced TNFα and IFNγ responses to M. leprae stimulation before and after 24 weeks treatment of LL with MDT and of ENL with prednisolone. The addition of autologous CD25+ cells to CD25+ depleted PBMCs abolished these responses. In both non-reactional LL and ENL groups mitogen (PHA)-induced TNFα and IFNγ responses were not affected by depletion of CD25+ cells either before or after treatment. Depleting CD25+ cells did not affect the IL-10 response to M. leprae before and after 24 weeks of MDT in participants with LL. However, depletion of CD25+ cells was associated with an enhanced IL-10 response on stimulation with M. leprae in untreated participants with ENL and reduced IL-10 responses in treated individuals with ENL. The enhanced IL-10 in untreated ENL and the reduced IL-10 response in prednisolone treated individuals with ENL was abolished by addition of autologous CD25+ cells. CONCLUSION: The findings support the hypothesis that the impaired cell-mediated immune response in individuals with LL is M. leprae antigen specific and the unresponsiveness can be reversed by depleting CD25+ cells. Our results suggest that the suppressive function of Tregs in ENL is intact despite ENL being associated with reduced numbers of Tregs. The lack of difference in IL-10 response in control PBMCs and CD25+ depleted PBMCs in individuals with LL and the increased IL-10 response following the depletion of CD25+ cells in individuals with untreated ENL suggest that the mechanism of immune regulation by Tregs in leprosy appears independent of IL-10 or that other cells may be responsible for IL-10 production in leprosy. The present findings highlight mechanisms of T cell regulation in LL and ENL and provide insights into the control of peripheral immune tolerance, identifying Tregs as a potential therapeutic target.

Development of a questionnaire to determine the case detection delay of leprosy: A mixed-methods cultural validation study.

BACKGROUND: Delay in case detection is a risk factor for developing leprosy-related impairments, leading to disability and stigma. The objective of this study was to develop a questionnaire to determine the leprosy case detection delay, defined as the period between the first signs of the disease and the moment of diagnosis, calculated in total number of months. The instrument was developed as part of the PEP4LEP project, a large-scale intervention study which determines the most effective way to implement integrated skin screening and leprosy post-exposure prophylaxis with a single-dose of rifampicin (SDR-PEP) administration in Ethiopia, Mozambique and Tanzania. METHODOLOGY/PRINCIPAL FINDINGS: A literature review was conducted and leprosy experts were consulted. The first draft of the questionnaire was developed in Ethiopia by exploring conceptual understanding, item relevance and operational suitability. Then, the first draft of the tool was piloted in Ethiopia, Mozambique and Tanzania. The outcome is a questionnaire comprising nine questions to determine the case detection delay and two annexes for ease of administration: a local calendar to translate the patient's indication of time to number of months and a set of pictures of the signs of leprosy. In addition, a body map was included to locate the signs. A 'Question-by-Question Guide' was added to the package, to provide support in the administration of the questionnaire. The materials will be made available in English, Oromiffa (Afaan Oromo), Portuguese and Swahili via https://www.infolep.org. CONCLUSIONS/SIGNIFICANCE: It was concluded that the developed case detection delay questionnaire can be administered quickly and easily by health workers, while not inconveniencing the patient. The instrument has promising potential for use in future leprosy research. It is recommended that the tool is further validated, also in other regions or countries, to ensure cultural validity and to examine psychometric properties like test-retest reliability and interrater reliability.

Evidence for hidden leprosy in a high leprosy-endemic setting, Eastern Ethiopia: The application of active case-finding and contact screening.

Leprosy or Hansen's disease is a disabling infectious disease caused by Mycobacterium leprae. Reliance on the self-presentation of patients to the health services results in many numbers of leprosy cases remaining hidden in the community, which in turn results in a longer delay of presentation and therefore leading to more patients with disabilities. Although studies in Ethiopia show pockets of endemic leprosy, the extent of hidden leprosy in such pockets remains unexplored. This study determined the magnitude of hidden leprosy among the general population in Fedis District, eastern Ethiopia. A community-based cross-sectional study was conducted in six randomly selected leprosy-endemic villages in 2019. Health extension workers identified study participants from the selected villages through active case findings and household contact screening. All consenting individuals were enrolled and underwent a standardized physical examination for diagnosis of leprosy. Overall, 262 individuals (214 with skin lesions suspected for leprosy and 48 household contacts of newly diagnosed leprosy cases) were identified for confirmatory investigation. The slit skin smear technique was employed to perform a bacteriological examination. Data on socio-demographic characteristics and clinical profiles were obtained through a structured questionnaire. Descriptive statistics and binary logistic regression were used to assess the association between the outcome variable and predictor variables, and the P-value was set at 0.05. From the 268 individuals identified in the survey, 6 declined consent and 262 (97.8%) were investigated for leprosy. Fifteen cases were confirmed as leprosy, giving a detection rate of 5.7% (95%, CI: 3%, 9%). The prevalence of hidden leprosy cases was 9.3 per 10,000 of the population (15/16107). The majority (93.3%) of the cases were of the multi-bacillary type, and three cases were under 15 years of age. Three cases presented with grade II disability at initial diagnosis. The extent of hidden leprosy was not statistically different based on their sex and contact history difference (p > 0.05). High numbers of leprosy cases were hidden in the community. Active cases findings, and contact screening strategies, play an important role in discovering hidden leprosy. Therefore, targeting all populations living in leprosy pocket areas is required for achieving the leprosy elimination target.

PEP4LEP study protocol: integrated skin screening and SDR-PEP administration for leprosy prevention: comparing the effectiveness and feasibility of a community-based intervention to a health centre-based intervention in Ethiopia, Mozambique and Tanzania.

INTRODUCTION: Leprosy, or Hansen's disease, remains a cause of preventable disability. Early detection, treatment and prevention are key to reducing transmission. Post-exposure prophylaxis with single-dose rifampicin (SDR-PEP) reduces the risk of developing leprosy when administered to screened contacts of patients. This has been adopted in the WHO leprosy guidelines. The PEP4LEP study aims to determine the most effective and feasible method of screening people at risk of developing leprosy and administering chemoprophylaxis to contribute to interrupting transmission. METHODS AND ANALYSIS: PEP4LEP is a cluster-randomised implementation trial comparing two interventions of integrated skin screening combined with SDR-PEP distribution to contacts of patients with leprosy in Ethiopia, Mozambique and Tanzania. One intervention is community-based, using skin camps to screen approximately 100 community contacts per leprosy patient, and to administer SDR-PEP when eligible. The other intervention is health centre-based, inviting household contacts of leprosy patients to be screened in a local health centre and subsequently receive SDR-PEP when eligible. The mobile health (mHealth) tool SkinApp will support health workers' capacity in integrated skin screening. The effectiveness of both interventions will be compared by assessing the rate of patients with leprosy detected and case detection delay in months, as well as feasibility in terms of cost-effectiveness and acceptability. ETHICS AND DISSEMINATION: Ethical approval was obtained from the national ethical committees of Ethiopia (MoSHE), Mozambique (CNBS) and Tanzania (NIMR/MoHCDEC). Study results will be published open access in peer-reviewed journals, providing evidence for the implementation of innovative leprosy screening methods and chemoprophylaxis to policymakers. TRIAL REGISTRATION NUMBER: NL7294 (NTR7503).

Knowledge of and Attitude Toward Leprosy in a Leprosy Endemic District, Eastern Ethiopia: A Community-Based Study.

INTRODUCTION: Leprosy or Hansen's disease is a potentially disabling disease that results in discrimination and self-stigma. A delay in case detection among leprosy patients is one of the factors resulting in disability. Although poor insights of the community toward leprosy lead to delays in case detection, studies on such matters are neglected in Ethiopia. OBJECTIVE: To assess the level of community knowledge and attitudes toward leprosy in Fedis District, Eastern Ethiopia. METHODS: A community-based cross-sectional study was carried out among 728 randomly selected households from July to August 2019. Each participant was interviewed using a pretested structured questionnaire consisting of participants' socio-demographic background, questions related to knowledge of and attitudes toward leprosy. The collected data were entered using EpiData 3.1 and analyzed using STATA version 13. Chi-squared test, binary, and multivariable logistic regressions were applied as appropriate to assess the association between outcome and independent variables. RESULTS: Among 728 study participants, 608 (83.52%) of them had heard about leprosy. Among the study participants who had heard of leprosy, 346 (56.91%) of them had high knowledge of leprosy. Multivariable logistic regression revealed that study participants who completed grade 1-8 (AOR=1.68, 95% CI=1.09-2.58, P=0.017) and government employees (AOR=7.56, 95% CI=2.23-25.63, P=0.001) were significantly associated with high level of knowledge of leprosy. Out of 608 study participants who had heard of leprosy, only 248 (40.79%) had a favorable attitude toward leprosy. Study participants who completed grade 1-8 (AOR= 2.72, 95% CI=1.76-4.19, P= 0.000) and urban inhabitants (AOR=0.49, 95% CI=0.31-0.75, P= 0.032) were significantly associated with favorable attitude toward leprosy. Having high knowledge of leprosy was significantly associated with favorable attitudes toward leprosy. CONCLUSION: This study revealed unfavorable attitudes toward leprosy among the community. Having a high overall knowledge level on leprosy has been shown to support a favorable attitude toward leprosy.

Whole blood RNA signatures in leprosy patients identify reversal reactions before clinical onset: a prospective, multicenter study.

Early diagnosis of leprosy is challenging, particularly its inflammatory reactions, the major cause of irreversible neuropathy in leprosy. Current diagnostics cannot identify which patients are at risk of developing reactions. This study assessed blood RNA expression levels as potential biomarkers for leprosy. Prospective cohorts of newly diagnosed leprosy patients, including reactions, and healthy controls were recruited in Bangladesh, Brazil, Ethiopia and Nepal. RNA expression in 1,090 whole blood samples was determined for 103 target genes for innate and adaptive immune profiling by dual color Reverse-Transcription Multiplex Ligation-dependent Probe Amplification (dcRT-MLPA) followed by cluster analysis. We identified transcriptomic biomarkers associated with leprosy disease, different leprosy phenotypes as well as high exposure to Mycobacterium leprae which respectively allow improved diagnosis and classification of leprosy patients and detection of infection. Importantly, a transcriptomic signature of risk for reversal reactions consisting of five genes (CCL2, CD8A, IL2, IL15 and MARCO) was identified based on cross-sectional comparison of RNA expression. In addition, intra-individual longitudinal analyses of leprosy patients before, during and after treatment of reversal reactions, indicated that several IFN-induced genes increased significantly at onset of reaction whereas IL15 decreased. This multi-site study, situated in four leprosy endemic areas, demonstrates the potential of host transcriptomic biomarkers as correlates of risk for leprosy. Importantly, a prospective five-gene signature for reversal reactions could predict reversal reactions at least 2 weeks before onset. Thus, transcriptomic biomarkers provide promise for early detection of these acute inflammatory episodes and thereby help prevent permanent neuropathy and disability in leprosy patients.

Identification of a systemic interferon-γ inducible antimicrobial gene signature in leprosy patients undergoing reversal reaction.

Reversal reactions (RRs) in leprosy are characterized by a reduction in the number of bacilli in lesions associated with an increase in cell-mediated immunity against the intracellular bacterium Mycobacterium leprae, the causative pathogen of leprosy. To identify the mechanisms that contribute to cell-mediated immunity in leprosy, we measured changes in the whole blood-derived transcriptome of patients with leprosy before, during and after RR. We identified an 'RR signature' of 1017 genes that were upregulated at the time of the clinical diagnosis of RR. Using weighted gene correlated network analysis (WGCNA), we detected a module of 794 genes, bisque4, that was significantly correlated with RR, of which 434 genes were part of the RR signature. An enrichment for both IFN-γ and IFN-ß downstream gene pathways was present in the RR signature as well as the RR upregulated genes in the bisque4 module, including those encoding proteins of the guanylate binding protein (GBP) family that contributes to antimicrobial responses against mycobacteria. Specifically, GBP1, GBP2, GBP3 and GBP5 mRNAs were upregulated in the RR peripheral blood transcriptome, with GBP1, GBP2 and GBP5 mRNAs also upregulated in the RR disease lesion transcriptome. These data indicate that RRs involve a systemic upregulation of IFN-γ downstream genes including GBP family members as part of the host antimicrobial response against mycobacteria.

The effects of prednisolone treatment on serological responses and lipid profiles in Ethiopian leprosy patients with Erythema Nodosum Leprosum reactions.

BACKGROUND: Erythema nodosum leprosum (ENL) is a systemic inflammatory complication occurring mainly in patients with lepromatous leprosy (LL) and borderline lepromatous leprosy (BL). Prednisolone is widely used for treatment of ENL reactions. However, it has been reported that prolonged treatment with prednisolone increases the risk for prednisolone-induced complications such as osteoporosis, diabetes, cataract and arteriosclerosis. It has been speculated that perhaps these complications result from lipid profile alterations by prednisolone. The effects of extended prednisolone treatment on lipid profiles in ENL patients have not been studied in leprosy patients with ENL reactions. Therefore, in this study we conducted a case-control study to investigate the changes in lipid profiles and serological responses in Ethiopian patients with ENL reaction after prednisolone treatment. METHODS: A prospective matched case-control study was employed to recruit 30 patients with ENL and 30 non-reactional LL patient controls at ALERT Hospital, Ethiopia. Blood samples were obtained from each patient with ENL reaction before and after prednisolone treatment as well as from LL controls. The serological host responses to PGL-1, LAM and Ag85 M. leprae antigens were measured by ELISA. Total cholesterol (TC), triglyceride (TG), high density lipoprotein (HDL) and low density lipoprotein (LDL) were measured by spectrophotometric method. RESULTS: The host antibody response to M. leprae PGL-1, LAM and Ag85 antigens were significantly reduced in patients with ENL reactions compared to LL controls after treatment. Comparison between patients with acute and chronic ENL showed that host-response to PGL-1 was significantly reduced in chronic ENL after prednisolone treatment. Untreated patients with ENL reactions had low lipid concentration compared to LL controls. However, after treatment, both groups had comparable lipid profiles except for LDL, which was significantly higher in patients with ENL reaction. Comparison within the ENL group before and after treatment showed that prednisolone significantly increased LDL and HDL levels in ENL patients and this was more prominent in chronic ENL than in acute patients with ENL. CONCLUSION: The significantly increased prednisolone-induced LDL and TG levels, particularly in patients with chronic ENL reactions, is a concern in the use of prednisolone for extended periods in ENL patients. The findings highlight the importance of monitoring lipid profiles during treatment of patients to minimize the long-term risk of prednisolone-induced complications.

Evaluation of Immunodiagnostic Tests for Leprosy in Brazil, China and Ethiopia.

Leprosy remains persistently endemic in several low- or middle income countries. Transmission is still ongoing as indicated by the unabated rate of leprosy new case detection, illustrating the insufficiency of current prevention methods. Therefore, low-complexity tools suitable for large scale screening efforts to specifically detect M. leprae infection and diagnose disease are required. Previously, we showed that combined detection of cellular and humoral markers, using field-friendly lateral flow assays (LFAs), increased diagnostic potential for detecting leprosy in Bangladesh compared to antibody serology alone. In the current study we assessed the diagnostic performance of similar LFAs in three other geographical settings in Asia, Africa and South-America with different leprosy endemicity. Levels of anti-PGL-I IgM antibody (humoral immunity), IP-10, CCL4 and CRP (cellular immunity) were measured in blood collected from leprosy patients, household contacts and healthy controls from each area. Combined detection of these biomarkers significantly improved the diagnostic potential, particularly for paucibacillary leprosy in all three regions, in line with data obtained in Bangladesh. These data hold promise for the use of low-complexity, multibiomarker LFAs as universal tools for more accurate detection of M. leprae infection and different phenotypes of clinical leprosy.

Evaluation of Auramine O staining and conventional PCR for leprosy diagnosis: A comparative cross-sectional study from Ethiopia.

BACKGROUND: Diagnosis of leprosy mainly relies on clinical examination due to the inconsistent sensitivity and poor reproducibility of the current laboratory tests. Utilisation of alternative methods to the standard Ziehl Neelsen (ZN), Fite-Faraco (FF) and Haematoxylin and Eosin (H&E) staining procedures may eventually improve leprosy diagnosis. METHODOLOGY/PRINCIPAL FINDINGS: In this comparative study, the performance of the fluorescent Auramine O (AO) staining and polymerase chain reaction (PCR) was assessed with different skin samples using a combination of ZN, FF and H&E staining as the gold standard. AO, ZN, FF, H&E and PCR tests were performed on slit skin smears (SSS) and/or punch biopsies collected from 141 clinically confirmed leprosy cases and 28 non-leprosy skin samples. DNA was extracted from punch biopsies using two different methods with or without mechanical lysis. Sensitivities were 87.6%, 59.3% and 77% for H&E, ZN and FF, respectively, whereas it reached 65.5% and 77.9% for AO in SSS and tissue sections and 91.1% for PCR in tissue samples. Morover, samples with low bacillary index, sensitivity of AO staining (61.8%) was similar to FF (60%, p>0.05) and lower than PCR (86.6%, p<0.05). Sensitivity of PCR also increased (96.8%, p<0.05) when mechanical lysis was used during DNA extraction compared to enzymatic treatment alone (84.6%). CONCLUSIONS/SIGNIFICANCE: Our results showed that for diagnostic purposes, analysis of skin section is more sensitive than SSS, especially for samples with low bacillary load. AO staining on SSS and tissue sections was not significantly better than other routine diagnostic tests but considerably more user friendly. The sensitivity of PCR was higher than current standard methods and increased when combined with more efficient DNA extraction using mechanical and chemical lysis. Therefore, we recommend AO staining for the diagnosis of leprosy in lower health facilities such as health centres and district hospitals and PCR diagnosis at referral level and research centres.

The Effects of Prednisolone Treatment on Cytokine Expression in Patients with Erythema Nodosum Leprosum Reactions.

Erythema nodosum leprosum (ENL) is a systemic inflammatory complication occurring mainly in patients with lepromatous leprosy (LL) and borderline lepromatous leprosy. Prednisolone is widely used for treatment of ENL reactions but clinical improvement varies. However, there is little good in vivo data as to the effect of prednisolone treatment on the pro-inflammatory cytokines in patients with ENL reactions. As a result, treatment and management of reactional and post-reactional episodes of ENL often pose a therapeutic challenge. We investigated the effect of prednisolone treatment on the inflammatory cytokines TNF, IFN-γ, IL-1ß, IL-6, and IL-17 and the regulatory cytokines IL-10 and TGF-ß in the skin lesion and blood of patients with ENL and compared with non-reactional LL patient controls. A case-control study was employed to recruit 30 patients with ENL and 30 non-reactional LL patient controls at ALERT Hospital, Ethiopia. Blood and skin biopsy samples were obtained from each patient before and after prednisolone treatment. Peripheral blood mononuclear cells from patients with ENL cases and LL controls were cultured with M. leprae whole-cell sonicates (MLWCS), phytohemagglutinin or no stimulation for 6 days. The supernatants were assessed with the enzyme-linked immunosorbent assay for inflammatory and regulatory cytokines. For cytokine gene expression, mRNA was isolated from whole blood and skin lesions and then reverse transcribed into cDNA. The mRNA gene expression was quantified on a Light Cycler using real-time PCR assays specific to TNF, IFN-γ, IL-ß, TGF-ß, IL-17A, IL-6, IL-8, and IL-10. The ex vivo production of the cytokines: TNF, IFN-γ, IL-1ß, and IL-17A was significantly increased in untreated patients with ENL. However, IL-10 production was significantly lower in untreated patients with ENL and significantly increased after treatment. The ex vivo production of IL-6 and IL-8 in patients with ENL did not show statistically significant differences before and after prednisolone treatment. The mRNA expression in blood and skin lesion for TNF, IFN-γ, IL-1ß, IL-6, and IL-17A significantly reduced in patients with ENL after treatment, while mRNA expression for IL-10 and TGF-ß was significantly increased both in blood and skin lesion after treatment. This is the first study examining the effect of prednisolone on the kinetics of inflammatory and regulatory cytokines in patients with ENL reactions before and after prednisolone treatment. Our findings suggest that prednisolone modulates the pro-inflammatory cytokines studied here either directly or through suppression of the immune cells producing these inflammatory cytokines.

Phylogenomics and antimicrobial resistance of the leprosy bacillus Mycobacterium leprae.

Leprosy is a chronic human disease caused by the yet-uncultured pathogen Mycobacterium leprae. Although readily curable with multidrug therapy (MDT), over 200,000 new cases are still reported annually. Here, we obtain M. leprae genome sequences from DNA extracted directly from patients' skin biopsies using a customized protocol. Comparative and phylogenetic analysis of 154 genomes from 25 countries provides insight into evolution and antimicrobial resistance, uncovering lineages and phylogeographic trends, with the most ancestral strains linked to the Far East. In addition to known MDT-resistance mutations, we detect other mutations associated with antibiotic resistance, and retrace a potential stepwise emergence of extensive drug resistance in the pre-MDT era. Some of the previously undescribed mutations occur in genes that are apparently subject to positive selection, and two of these (ribD, fadD9) are restricted to drug-resistant strains. Finally, nonsense mutations in the nth excision repair gene are associated with greater sequence diversity and drug resistance.

Phylogenomics and antimicrobial resistance of the leprosy bacillus Mycobacterium leprae

Leprosy is a chronic human disease caused by the yet-uncultured pathogen Mycobacterium leprae. Although readily curable with multidrug therapy (MDT), over 200,000 new cases are still reported annually. Here, we obtain M. leprae genome sequences from DNA extracted directly from patients' skin biopsies using a customized protocol. Comparative and phylogenetic analysis of 154 genomes from 25 countries provides insight into evolution and antimicrobial resistance, uncovering lineages and phylogeographic trends, with the most ancestral strains linked to the Far East. In addition to known MDT-resistance mutations, we detect other mutations associated with antibiotic resistance, and retrace a potential stepwise emergence of extensive drug resistance in the pre-MDT era. Some of the previously undescribed mutations occur in genes that are apparently subject to positive selection, and two of these (ribD, fadD9) are restricted to drug-resistant strains. Finally, nonsense mutations in the nth excision repair gene are associated with greater sequence diversity and drug resistance.

New Insight into the Pathogenesis of Erythema Nodosum Leprosum: The Role of Activated Memory T-Cells.

Memory T-cells, particularly, effector memory T cells are implicated in the pathogenesis of inflammatory diseases and may contribute to tissue injury and disease progression. Although erythema nodosum leprosum (ENL) is an inflammatory complication of leprosy, the role of memory T cell subsets has never been studied in this patient group. The aim of this study was at investigate the kinetics of memory T cell subsets in patients with ENL before and after prednisolone treatment. A case-control study design was used to recruit 35 untreated patients with ENL and 25 non-reactional lepromatous leprosy (LL) patient controls at ALERT Hospital, Ethiopia. Venous blood samples were obtained before, during, and after treatment from each patient. Peripheral blood mononuclear cells (PBMCs) were isolated and used for immunophenotyping of T cell activation and memory T-cell subsets by flow cytometry. The kinetics of these immune cells in patients with ENL before and after treatment were compared with LL patient controls as well as within ENL cases at different time points. The median percentage of CD3+, CD4+, and CD8+ T-cells expressing activated T-cells were significantly higher in the PBMCs from patients with ENL than from LL patient controls before treatment. The median percentage of central and activated memory T-cells was significantly increased in patients with ENL compared to LL patient controls before treatment. Interestingly, patients with ENL had a lower percentage of naïve T cells (27.7%) compared to LL patient controls (59.5%) (P < 0.0001) before treatment. However, after prednisolone treatment, patients with ENL had a higher median percentage of naïve T-cells (43.0%) than LL controls (33.0%) (P < 0.001). The median percentage of activated T-cells (effector memory and effector T-cells) was significantly increased in patients with ENL (59.2%) before treatment compared to after treatment with prednisolone (33.9%) (P < 0.005). This is the first work which has shown T-cell activation and the different subsets of memory T cells in untreated patients with ENL. Consequently, this study delineates the role of T-cell activation in the pathogenesis of ENL reaction and challenges the long-standing dogma of immune complex as a sole etiology of ENL reaction.