Sensitivity of a Rapid Mix Test with Combined Synthetic Antigens Derived from Mycobacterium Leprae PGL-1 for Diagnosis and Surveillance of Leprosy

Introduction: In the Americas, Brazil contributes 91.63% of the total cases and the state of Pará still has high endemia for leprosy. Objective: To analyze the performance of a rapid test for the diagnosis and epidemiological surveillance of leprosy in endemic areas. Methods: The sample consisted of 70 MB multibacillary leprosy (MB) patients, 63 paucibacillary (PB) patients, and 80 intradomiciliary consanguineous contacts (ICSCO) of patients. A rapid test with a 15-minute reading was applied using two prototypes: prototype 1, double test with trisaccharide antigen (NT-P-BSA) at 1a. line (83.2 ng/test) and disaccharide antigen (ND-O-BSA) at 2a. (83.2 ng/test), both with a flow of 0.08 μL/mm with a 10 μC membrane, anti-IgM conjugate with a flow of 0.040 μL/mm and a Tris-Triton and prototype 2 runner buffer with MIX antigen (trisaccharide + disaccharide) in the same concentrations and conditions of prototype 1. Results: The comparison of the MIX test positivity rate and the disaccharide or trisaccharide doublet test across all samples was statistically significant, demonstrating that the MIX test had higher seropositivity rates compared to the ND-O-BSA or NT-P-BSA. It was demonstrated that the MIX test showed a good performance, with 25.39% of the PB patients negative for the disaccharide and trisaccharide duplet test, but positive for MIX. Conclusions: These data suggest the potential for further optimizing the performance by adding other synthetic antigens to the MIX antigens.


Introduction
Leprosy is an endemic disease prevalent in developing countries [1]. It is still associated with precarious living conditions and occurs in socially vulnerable populations [2] [3], which often have restricted access to health services. Scientific studies have sought to establish immunological diagnostic methods applicable to, and suitable for endemic regions with few technical-diagnostic resources [3].
The latex agglutination technique in the 1950s represented the beginning of the studies towards the production of immunochromatographic rapid reading tests. Subsequently, rapid tests were widely studied in the 1980s and finally established in the 1990s and 2000s [4] [5] [6] [7] [8]. The most well-known formats of immunochromatographic tests are the lateral flow immunochromatography test (ML Flow), double migration immunochromatography, and immunoconcentration, and solid phase devices [8]. The lateral flow immunochromatographic test detects anti-PGL-1 (phenolic-glycolipid-1) IgM antibodies. The sensitivity of the ML Flow test is 97.4% in patients with multibacillary leprosy (ML) and the specificity is 90.2%; it has an excellent correlation with ELISA data (91%; k = 0.77) [6].
The ML Flow test uses the trisaccharide antigen (NT-P-BSA), colloidal gold and conjugate containing anti-human IgM, dispensed into the test control line [4] [5] [6]. Another rapid test developed by the Infectious Disease Research (IDRI), the Institute of Infectious Diseases Research in the United States, in cooperation with the Brazilian company Orange Life [7] was approved by ANVISA (National Health Surveillance Agency) in 2012. The NDO-LID-1 test, which merges the two recombinant proteins ML0405 and ML 2331 together with the NDO antigen showed 93.3% specificity for both MB and PB patients. The sensitivity was 95.7% for the MB and 73.7% for the PB patients [7]. Therefore, it is important that these studies are carried out in countries where leprosy still represents a public health problem and leads to social and economic difficulties for the affected population, making it more vulnerable and increasing the burden on the state through the need of treatment and disability pensions.
The objective of this study was to determine the sensitivity of a rapid test with synthetic antigens derived from Mycobacterium leprae. PGL-1 combined, disaccharide antigen (ND-O-BSA) [4] was produced in North America and trisaccharide antigen (NT-P-BSA) [4] in Japan for the diagnosis and epidemiological surveillance of leprosy, especially of patients with paucibacillary leprosy (PB). These patients have a genetic-immunological profile expressing a cellular-based immune response and have low bacillary loads and milder symptoms. It is more challenging to define the clinical classification for specific treatment purposes in these cases [5]. Therefore, a rapid sensitivity test for such cases may be of great help to health professionals responsible for clinical diagnosis [4] [5].  Epi-Info 2000 and BioStat 5.0 software were used to calculate the Odds Ratio (OR) and determine the cut-off point, sensitivity and specificity of the screening test, the MIX rapid test and the Duplete test for the studied samples. In addition,  The screening test for the MB group using the MIX test compared to the ICSCO group for the ND-O-BSA test showed a sensitivity of 84.29%, a specificity of 93.75%, false-positive = 6.25%, false-negative = 15.71%; Prevalence = 0.467 or 46.67%; Predictive value of positive test (PPV) = 92.19%, negative predictive value (NPV) = 87.21%, accuracy = 0.89 or 89.33%, +LR Likelihood Ratio positive = 13.49, -LR Likelihood Negative ratio = 0.17 (Table 2). For the group of PB patients using the rapid MIX test compared to the CCOSI group using the ND-O-BSA test, a sensitivity of 46.03%, specificity of 93.75%, false-positive = 6.25%, false-negative = 53.97%, prevalence = 0.441 or 44.06%, PPV = 85.29%, NPV = 68.81%, accuracy = 0.73% or 72.73%, +positive LR = 7.37, and -LR negative = 0.58. The comparison between PB patients and the ICSCO group using the ND-O-BSA test showed a sensitivity of 12.70%, specificity of 93.75%, false-positive = 6.25%, false-negative = 87.30%, prevalence = 0.441 or 44.06%, PPV = 61.54%, NPV = 57.69%, accuracy = 0.58 or 58.04%, +LR = 2.03%, -LR = 0.93 ( Table 3).

Methods
The comparison between the Mix test and the ND-O-BSA test in the PB group showed a probability of seropositivity of 26.01, p < 0.0001 (95% CI-5.84 ≤ μ ≤ 115.78) for the MIX test, a sensitivity of 46.03% and a specificity of 93.75% (Table 1 and Table 4). The comparison between the Mix test and the NT-P-BSA test for the probability of seropositivity for the MIX test was 5.86, p < 0.0001 (95% CI -2.4 μM ≤ 14.30), a sensitivity of 46.03% and a specificity of 87.30% (Table 1).

Discussion
Epidemiological studies using immunological and molecular methods have been   [6], which uses the trisaccharide antigen. Moreover, in PB Accordingly, we demonstrated that the MIX test showed a good performance, with 25.39% of the PB patients negative for the disaccharide and trisaccharide duplet test, but positive for MIX. These data suggest the potential for further optimizing the performance by adding other synthetic antigens to the MIX antigens.

Conclusions
The rapid MIX test with combined M. leprae antigens proved to be an excellent tool for the diagnosis and surveillance of leprosy in endemic areas, compared to the rapid double-disaccharide and/or trisaccharide test. In this study, the need to improve the performance of the MIX test, adding other synthetic antigens was determined. This may reduce the false negative rate and improve the diagnosis of multibacillary leprosy, especially paucibacillary leprosy.