Edited by: Juarez Antonio Simões Quaresma, Universidade do Estado do Pará, Brazil
Reviewed by: Mayara Ingrid Sousa Lima, Federal University of Maranhão, Brazil; Tinara Leila De Souza Aarão, Federal University of Pará, Brazil
This article was submitted to Pathology, a section of the journal Frontiers in Medicine
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Hansen’s disease (HD) is an ancient disease, but more than 200,000 new cases were reported worldwide in 2019. Currently, there are not many satisfactory immunoassay methods for its diagnosis. We evaluated antibodies against Mce1A as a promising new serological biomarker. We collected plasma from new cases, contacts, and endemic controls in the city of Parnaíba and treated patients at Carpina, a former HD colony in Piauí state, northeastern Brazil. Receiver operating characteristic (ROC) curves were used to assess the assay thresholds, specificity and sensitivity of the IgA, IgM, and IgG antibodies against α-Mce1A by indirect ELISA and compared it with IgM anti-PGL-I and molecular diagnosis by quantitative polymerase chain reaction (qPCR). Venn diagrams were generated to represent the overlap in the antibody positivity pattern. Multivariate analysis was performed to assess the potential predictor of antibodies for the outcome of having an HD diagnosis. IgA and IgG were positive in 92.3 and 84% of patients, respectively. IgM was negative for all treated patients. IgG had a sensitivity and specificity of 94.7 and 100%, respectively. IgM-positive individuals had a 3.6 chance of being diagnosed with HD [OR = 3.6 (95% CI = 1.1–11.6);
HD is a disabling chronic infectious disease caused by
The strategy of an early diagnosis and effective treatment with multidrug therapy (MDT) is crucial for HD cure, preventing sequelae and reducing the disease stigma (
There is no laboratory test capable of detecting all clinical forms of HD. The knowledge and skills required for an HD diagnosis, treatment and management by general health workers are unsatisfactory, leading to delayed diagnosis, physical disabilities, socioeconomic impairment, and continued
However, there is a need for simple, low-cost diagnostic strategies to monitor treatment and assess household transmissions at primary care settings. The mammalian cell-entry 1A (Mce1A) protein, first described in
A cross-sectional study was conducted at the National Reference Center in Sanitary Dermatology and HD, Clinical Hospital of Ribeirão Preto Medical School (HCFMRP-USP), University of São Paulo, which provides training in HD management for several states of Brazil (MH-Brazil Project). Volunteer subjects were recruited by convenience sampling in March 2016 during a campaign to evaluate contacts of patients in the city Parnaíba, Brazilian municipality in the state of PI, the second most populous city in the state. Treated HD patients living in a former HD colony (Colony of Carpina), PI, Brazil, were also included in this study.
After signing an informed consent form, the volunteers were classified into four groups: (1) new cases of HD diagnosed during active search actions in the Parnaíba Municipality (PAR-NC), (2) treated HD patients who were residents of the Carpina Colony Hospital (CAR-TP), (3) household contacts (HHC) evaluated in Parnaíba, and (4) healthy endemic controls (EC) (
Study population characteristics (
EC |
HHC |
PAR-NC |
CAR-TP |
||
Age, years, mean (SD) | 29.5 (12.3) | 42.8 (16.7) | 43.9 (16.9) | 58.6 (13.0) | <0.0001 |
Male | 7 (35) | 9 (52.9) | 17 (65.4) | 15 (78.9) | 0.03 |
Female | 13 (65) | 8 (47.1) | 9 (34.6) | 4 (21.1) | |
PB | − | − | 2 (7.7) | 1 (5.3) | 0.009 |
MB | − | − | 24 (92.3) | 10 (52.6) | |
DDS | − | − | 0 (0) | 5 (26.3) | |
I | − | − | 1 (3.8) | − | |
TT | − | − | 1 (3.8) | − | |
BT | − | − | 1 (3.8) | − | |
BB | − | − | 13 (50) | − | |
BL | − | − | 3 (11.6) | − | |
LL | − | − | 7 (27) | − | |
Positive | − | − | 15 (57.7) | 11 (57.9) | 0.09 |
Negative | − | − | 3 (11.5) | 8 (42.1) | |
Ct, mean (SD) | − | − | 28⋅07 (1.065) | 30⋅96 (0.330) | 0.03 |
Newly diagnosed HD cases seen at Parnaíba and Carpina were invited to participate in this study. Patients were considered eligible for inclusion in the study if their diagnosis was confirmed by clinical evaluation and serological and/or molecular exams. All cases were classified considering the guidelines adapted by Indian Association of Leprologists (
HHC was defined as volunteers residing in the same household with an index case for at least 6 months prior to diagnosis. All HHC and EC were clinically screened for signs or symptoms suggestive of HD and subjected to laboratory analysis with serological examination. Clinical examinations were performed by trained physicians and health professionals at HCFMRP-USP.
ECs, representing community contacts, were defined as healthy individuals residing in the city Parnaiba, PI, Brazil an endemic area who had no history of diagnosis or contact with an HD. All participants reported being test-negative for the human immunodeficiency virus and did not diseases or use immunosuppressive drugs.
Indirect enzyme-linked immunosorbent assay (ELISA) was used to measure the anti-PGL-I IgM titer of every serum sample according to a previously reported protocol (
Total DNA extraction of earlobes and at least one elbow and/or lesion SSS sample was performed with the QIAamp DNA Mini Kit (Qiagen, Germantown, MD, cat: 51306) was performed according to the manufacturer’s protocol. DNA was used to perform PCR-RLEP according to a previously reported protocol (
Quantitative assessment of IgA, IgM and IgG antibodies against the Mce1A protein was performed by indirect ELISA (
All data were analyzed by GraphPad Prism v. 9.0 software (GraphPad Inc., La Jolla, CA, United States). Statistical variations were analyzed by the Mann–Whitney and Kruskal–Wallis tests, followed by Dunn’s test. Spearman’s correlation was used to compare the immunoglobulin levels of IgA, IgM, IgG anti-Mce1A and IgM anti-PGL-I. The ability of immunoglobulin levels to discriminate HD patients from controls (EC) was evaluated by receiver operating characteristic (ROC) curves. The chi-square test was used to assess associations among categorical variables and the presence of antibodies. Comparisons of the qPCR-RLEP positive test results were performed by the
The funder of the study had no role in the study design, data collection, data analysis, data interpretation, or writing of the report. All authors had full access to all of the data in the study and had final responsibility for the decision to submit for publication.
The study included 82 volunteers, grouped as new HD patients from Parnaíba (PAR-NC;
The antibody profiles against Mce1A protein and PGL-I levels in PAR-NC, CAR-TP, HHC, and EC are shown in
Antibodies anti-Mce1a are biomarkers for the diagnosis and monitoring of HD. IgA
A panel comprising plasma samples from PAR-NC, CAR-TP and HHC was examined (
Comparison of receiver operating characteristic curve analysis for IgA, IgM, and total IgG against Mce1A protein and IgM anti-PGL-I in discriminating among new HD patients, treated HD patients and HHC vs. endemic controls.
Group | Antibody | AUC (95%CI) | Cut-off |
Sensitivity% (95%CI) | Specificity% (95%CI) | LR + | |
IgA | 0.95 | <0.0001 | 0.203 | 93.2 (74.8–99.0) | 88.2 (72.5–96.7) | 7.8 | |
IgM | 0.83 | <0.0001 | 0.184 | 57.6 (36.9–76.6) | 85.0 (62.1–96.7) | 3.8 | |
PAR-NC | IgG | 0.97 | <0.0001 | 0.302 | 88.4 (69.8–97.5) | 100 (83.1–100) | − |
APGL-I | 0.81 | 0.0002 | 0.295 | 38.4 (20.2–59.4) | 100 (83.1–100) | − | |
IgA | 0.80 | 0.0012 | 0.203 | 52.6 (28.8–75.5) | 100 (83.1–100) | − | |
IgM | 0.60 | 0.27 | 0.184 | 100 (82.3–100) | 15 (3.2–37.8) | 1.1 | |
CAR-TP | IgG | 0.99 | <0.0001 | 0.302 | 94⋅7 (73.9–99.8) | 100 (83.1–100) | − |
APGL-I | 0.60 | 0.26 | 0.295 | 10.5 (1.3–33.1) | 100 (83.1–100) | − | |
IgA | 0.92 | <0.0001 | 0.203 | 82.3 (56.5–96.2) | 100 (83.2–100) | − | |
IgM | 0.64 | 0.12 | 0.184 | 58.8 (32.9–81.5) | 60 (36.0–80.8) | 1.4 | |
HHC | IgG | 0.96 | <0.0001 | 0.302 | 88.2 (63.5–98.5) | 100 (83.1–100) | − |
APGL-I | 0.67 | 0.06 | 0.295 | 11.7 (1.4–36.4) | 100 (83.1–100) | − |
ELISA performance of anti-Mce1A antibody levels. Receiver operating characteristic analysis for comparison of IgA anti-Mce1A
The IgA titer was positive in 92.3% of PAR-NC patients, and 84% were positive for IgG, regardless of the clinical and operational classification of the evaluated cases. The Anti-Mce1A IgM titer was positive in 50% of HD patients, while the positive APGL-I titer was detected only in 38.5%. In contrast, treated patients had no detection of anti-Mce1A IgM antibodies, and only 11.1% were positive for APGL-I. IgA and IgG titers remained positive in 52.6 and 89.5% of cases, respectively. Among the HHC, IgA and IgG antibody titers were positive in 88.2 and 64.7% of patients, respectively. The IgM titer among HHC was positive in 29.4% [index median: 0.8 (IQR: 0.6–1.04)], 17.6% better than APGL-I [index median: 0.4 (IQR: 0.1–0.8)] (
Positivity to antibodies against Mce1A protein and PGL-I in different groups of studies.
Groups | No of cases | IgA |
IgM |
IgG |
APGL-I |
||||
PAR-NC | 26 | 24 (92.3) | 34.7;<0.0001 | 13 (50) | 6.1; 0.01 | 22 (84.6) | 32.4;<0.0001 | 10 (38.5) | 9.8; 0.001 |
CAR-TP | 19 | 10 (52.6) | 10.9; 0.0009 | 0 (0) | 3.0; 0.07 | 17 (89.5) | 31.7;<0.0001 | 2 (11.1) | 2.2; 0.1 |
HHC | 17 | 15 (88.2) | 25.9;<0.0001 | 5 (29.4) | 1.1; 0.2 | 11 (64.7) | 18.4<0.0001 | 2 (11.8) | 2.4; 0.1 |
EC | 20 | 1 (5.0) | 3 (15) | 0 (0) | 0 (0) |
The antibody titers differed for the three study groups (PAR-NC, CAR-TP, and HHC), considering that newly diagnosed patients (PAR-NC) had a greater number of positive individuals for all biomarkers tested (IgA, IgM, IgG, and APGL-I) with a predominance of positive anti-Mce1A IgM titers. Treated patients (CAR-TP) had positive titers only for IgA and IgG anti-Mce1A antibodies. APGL-I antibodies did not achieve a satisfactory response for detecting HD cases and infected HHC (
Seropositivity pattern of antibodies in HD. The Venn diagrams represent the overlap in the number of positive IgA, IgM, IgG anti-Mce1A, and APGL-I antibodies in each of the comparison groups new cases of HD in Parnaíba (PAR-NC), treated HD patients in Colony Carpina (CAR-TP) and household contacts of HD patients in Parnaíba (HHC).
Correlation analyses were performed to assess the different levels of the serological markers tested. There was a strong positive correlation between the indices of anti-Mce1A IgA and IgG in the PAR-HD group (
Immunoglobulins correlation profile in HD. Spearman’s correlation coefficient was used to compare immunoglobulin indices. PAR-NC new cases of HD in Parnaíba
The logistic regression model demonstrated an association of IgM, IgA and PCR-RLEP with the clinical outcomes. (
Potential of anti-Mce1A antibodies as predictors of the diagnosis of HD. Binomial logistic regression analyses showing odds ratios (ORs) and 95% confidence intervals (CIs) for HD diagnosis with independent variables: age, sex, PCR-RLEP, and antibodies against Mce1A (IgA, IgM, and IgG) for all groups (PAR-NC, CAR-TP, HHC, and EC)
HD patients were compulsorily institutionalized in HD colonies before the establishment of specific treatment. Due to late diagnosis, insufficient treatment, or patients initially highly infected, colony cases of HD had a high bacillary load at diagnosis, who remained test positive by PCR in 57.9% of treated patients. Large numbers of dead
The levels of IgA and IgG antibody titers against the Mce1A protein in the three groups we evaluated (contacts, new cases and treated patients) were significantly elevated but not among the healthy controls in endemic communities. IgM appeared to be a sensitive biomarker for identifying active disease, since no colony population treated with MDT had a positive titer. Positive IgM titer indicates a need for a robust clinical investigation of HHCs and individuals in endemic regions for HD.
The serological assays using PGL-I or LID antigens (NDO-BSA, NDO-LID, LID-1, and others) already reported present results in the literature with high seropositivity almost only in MB patients. However, anti-Mce1A antibodies demonstrate satisfactory seropositivity for the antibodies tested in both PB and MB patients and even in household contacts, as a complementary diagnostic tool capable of detecting potential cases early, as previously described by Lima et al. (
The positive anti-Mce1A IgG antibody titers (89.5%) with high diagnostic accuracy (94.7% sensitivity and 100% specificity) and negative anti-Mce1A IgM antibody titers (0%) among treated HD patients in the colony indicate the potential of these serological markers to monitor treatment response.
This group’s previous work in a population from another endemic region in the state of Bahia showed elevated IgA, IgM, and IgG titers in cases of paucibacillary and multibacillary HD. BCG vaccination and latent tuberculosis infection did not induce cross-reactive anti-Mce1A antibodies in HD patients (
The antibody response to PGL-I is the most widely evaluated biomarker for HD, and it has been shown extensively that the detection of α-PGL-I antibodies only is not sufficient to identify all HD patients, and PB cases generally lack an antibody response against PGL-I (
Summarizing and aggregating the results from our three previous published studies searching for HD involving prison male/female populations and from the community (
Several tests have been developed to assess anti-PGL-I antibody, a known biomarker of
Therefore, compared to the traditional clinical and other laboratory tests for HD, the anti-Mce1A serology results proved to be superior for the diagnosis of new cases of HD (including PB cases), monitoring treatment response, and identifying infected HHC of index cases.
Brazilian epidemiological indicators and the current global HD situation confirm the scenario of continued transmission and its maintenance as a public health problem that has not yet been resolved. The reality of the disease in Brazil reinforces the importance of developing new tools for the diagnosis and monitoring of HD, and studies that address the humoral immunological profile of HD patients and their contacts in addition to anti-PGL-I are rare, with results limited to multibacillary only and which is still being implemented in public health services in the country. Therefore, the development of HD diagnostic techniques in all clinical forms, both multi and paucibacillary and/or as a way of monitoring cases, in addition to their contacts and the expected search for an early diagnosis in its subclinical phase become goals to be sought to achieve the HD control goals recommended by the WHO and intended by the Ministry of Health.
In summary, in addition to understanding of the role of Mce1A in the pathogenesis of HD, it offers a highly useful target for immunological biomarker response for the implementation strategies of low-cost and easy-to-perform serological diagnostic platforms for HD. Such platforms will constitute an important technological advance for public health control of HD that can interrupt the chain of transmission of the disease, in addition to preventing deformities, disability and stigma associated with this ancient disease.
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.
The studies involving human participants were reviewed and approved by the Institutional Review Board for Human Research of the HCFMRP-USP (MH-Brazil Project—Protocol number 16620/2014). Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.
FL and MF substantially contributed to manuscript conception and design, acquisition of data, and analysis and interpretation of data. MF, JS, RA, and EA contributed to the clinical care of patients and collected the samples. FL, VA, VB, and NP contributed to the development of experiments. FL and DT contributed to the statistical analysis and interpretation of the data. LR and SA conducted scientific guidance and advice. MF gave final approval of the final submitted version. All authors contributed to the article and approved the submitted version.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brazil (CAPES)—Finance Code 001, the National Council for Scientific and Technological Development (CNPq) with Ph.D. scholarships program for FL and Unified Scholarships Program (PUB-USP for VA. Brazilian Health Ministry (MS/FAEPA-FMRP-USP: 749145/2010 and 767202/2011) and Oswaldo Cruz Foundation—Ribeirão Preto (TED 163/2019–Process: N° 25380.102201/2019-62/Project Fiotec: PRES-009-FIO-20) with financial support to the National Referral Center for Sanitary Dermatology and HD and Research and Assistance Support Foundation of the Hospital of the Medical School of Ribeirão Preto at USP (FAEPA).
We thank the staff members and all patients who agreed to participate in this study. The following reagent was obtained through BEI Resources, NIAID, NIH: PGL-I ND-O-BSA.