Host immune responses induced by specific Mycobacterium leprae antigens in an overnight whole-blood assay correlate with the diagnosis of paucibacillary leprosy patients in China

Abstract

Background Leprosy, caused by Mycobacterium leprae , affects over 200,000 people annually worldwide and remains endemic in the ethnically diverse, mountainous and underdeveloped southwestern provinces of China. Delayed diagnosis of leprosy persists in China, thus, additional knowledge to support early diagnosis, especially early diagnosis of paucibacillary (PB) patients, based on the host immune responses induced by specific M . leprae antigens is needed. The current study aimed to investigate leprosy patients and controls in Southwest China by comparing supernatants after stimulation with specific M . leprae antigens in an overnight whole-blood assay (WBA) to determine whether host markers induced by specific M . leprae antigens improve the diagnosis or discrimination of PB patients with leprosy. Methodology/Principal findings Leprosy patients [13 multibacillary (MB) patients and 7 PB patients] and nonleprosy controls [21 healthy household contacts (HHCs), 20 endemic controls (ECs) and 19 tuberculosis (TB) patients] were enrolled in this study. The supernatant levels of ten host markers stimulated by specific M . leprae antigens were evaluated by overnight WBA and multiplex Luminex assays. The diagnostic value in PB patients and ECs and the discriminatory value between PB patients and HHCs or TB patients were evaluated by receiver operator characteristics (ROC) analysis. ML2044-stimulated CXCL8/IL-8 achieved the highest sensitivity of 100%, with a specificity of 73.68%, for PB diagnosis. Compared to single markers, a 3-marker combination model that included ML2044-induced CXCL8/IL-8, CCL4/MIP-1 beta, and IL-6 improved the diagnostic specificity to 94.7% for PB patients. ML2044-stimulated IL-4 and CXCL8/IL-8 achieved the highest sensitivity (85.71% and 100%) and the highest specificity (95.24% and 84.21%) for discriminating PB patients from HHCs and TB patients, respectively. Conclusions Our findings suggest that the host markers induced by specific M . leprae antigens in an overnight WBA increase diagnostic and discriminatory value in PB patients with leprosy, with a particularly strong association with interleukin 8.

Transcriptomic Analysis of Mycobacterium leprae-Stimulated Response in Peripheral Blood Mononuclear Cells Reveal Potential Biomarkers for Early Diagnosis of Leprosy

“…IL-8 production is significantly increased in the PBMCs from patients with erythema nodosum leprosum in vitro when compared to those of lepromatous leprosy controls (Negera et al, 2018). Our previous study with Luminex technology has also found that IL-8 cytokine expression is elevated in leprosy patients upon stimulation with M. leprae antigen ML2044 (Chen et al, 2019), which confirms our findings at the transcriptomic level in this study. Thus, our finding of increased IL-8 mRNA/gene expression in the blood from leprosy patients is consistent with a potential role for this chemokine in the pathogenesis of erythema nodosum leprosum in leprosy (Negera, et al, 2018;Bhat and Vaidya, 2020).…”

“…Therefore, screening novel M. leprae-specific antigens, combining different M. leprae antigens, and a multi-cytokine analytic model is needed for a more effective diagnosis of leprosy. Many studies have identified signature genes or proteins using whole-blood assay and PBMCs, which could distinguish between leprosy patients, ECs, and HHCs (Sampaio et al, 2011;Geluk et al, 2012;Oliveira et al, 2014;Freitas et al, 2016;Hungria et al, 2017;Chen et al, 2018;Chen et al, 2019). However, the relationship between transcriptome features useful for early diagnosis of leprosy is seldom discussed (Tio-Coma et al, 2019).…”

“…Definitive diagnosis of leprosy by clinic and pathological features requires experienced physicians. Although the host biomarkers with a positive immune response to M. leprae through whole-blood assay have potential diagnostic value for leprosy, especially for paucibacillary (PB) patients (Chen et al, 2019;Geluk et al, 2010;Hungria et al, 2017;Sampaio et al, 2011;van Hooij and Geluk, 2021), there is currently no diagnostic test based on transcriptomic analysis of leprosy patients in China. Therefore, the development of an accurate clinical diagnostic test is urgently needed.…”

Prevention and Treatment of Leprosy — China, 2009−2019

“…The Beijing Tropical Medicine Research Institute (BTMRI) evaluated the ability of M. leprae antigen specific immune responses to support the leprosy diagnosis ( 14 - 17 ). In addition, BTMRI tested a panel of M. leprae -stimulated host markers in an overnight whole-blood assay and found that M. leprae -induced CXCL8/IL-8 showed potential diagnostic and discriminatory value for PB patients ( 18 ). These simple tools could be set up in endemic areas without clinical molecular laboratories.…”

Blood RNA signature RISK4LEP predicts leprosy years before clinical onset

“…In the past years, several studies have searched for biomarkers to (early) detect leprosy either based on the host immune response [26] , [27] , [28] , [29] , [30] , [31] , the pathogen [32] , [33] , [34] , [35] , [36] , [37] , or a combination of both [38] , [39] , [40] , [41] , [42] , [43] , [44] , [45] . Molecular detection by identification of the repetitive element RLEP by (quantitative) PCR [ 33 , 46 , 47 ] as well as detection of anti- M. leprae phenolic glycolipid I (PGL-I) IgM in blood [ 28 , 29 ] are methods employed to assist leprosy diagnosis.…”